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Sample records for differentiated pc12 cells

  1. Expression of dystrophin Dp71 during PC12 cell differentiation.

    PubMed

    Cisneros, B; Rendon, A; Genty, V; Aranda, G; Marquez, F; Mornet, D; Montañez, C

    1996-08-02

    The expression of dystrophin-protein 71 (Dp71) was investigated during nerve growth factor (NGF) induced differentiation of PC12 cells. A semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was designed to measure Dp71 mRNA, whereas the Dp71 protein amount was evaluated by immunoblot analysis using an anti-dystrophin monoclonal antibody. Comparison with control cultures showed that Dp71 mRNA and protein levels increased in parallel with NGF treatment peaking with increments of 60% and 1.4 times, respectively. The upregulation of Dp71 expression during PC12 cells differentiation point at PC12 cells as a suitable model for studying the function of Dp71 in neuronal cells.

  2. Effect of Cuscuta chinensis glycoside on the neuronal differentiation of rat pheochromocytoma PC12 cells.

    PubMed

    Jian-Hui, Liu; Bo, Jiang; Yong-Ming, Bao; Li-Jia, An

    2003-08-01

    Exposure of rat pheochromocytoma PC12 cells to Cuscuta chinensis glycoside induced neuronal differentiation with resulting outgrowth of neurites and increase of acetylcholinesterase activity. A specific inhibitor of mitogen-activated protein kinase (MAPK) kinase, PD98059, prevented this effect of C. chinensis on PC12 cells. These results suggested that C. chinensis glycoside induced neuronal differentiation in PC12 cells linked to the mitogen-activated protein kinase signaling cascade.

  3. PC12 Cells Differentiate into Chromaffin Cell-Like Phenotype in Coculture with Adrenal Medullary Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Mizrachi, Yaffa; Naranjo, Jose R.; Levi, Ben-Zion; Pollard, Harvey B.; Lelkes, Peter I.

    1990-08-01

    Previously we described specific in vitro interactions between PC12 cells, a cloned, catecholamine-secreting pheochromocytoma cell line derived from the rat adrenal medulla, and bovine adrenal medullary endothelial cells. We now demonstrate that these interactions induce the PC12 cells to acquire physical and biochemical characteristics reminiscent of chromaffin cells. Under coculture conditions involving direct cell-cell contact, the endothelial cells and the PC12 cells reduced their rates of proliferation; upon prolonged coculture PC12 cells clustered into nests of cells similar to the organization of chromaffin cells seen in vivo. Within 3 days in coculture with endothelial cells, but not with unrelated control cells, PC12 cells synthesized increased levels of [Met]enkephalin. In addition, PC12 cells, growing on confluent endothelial monolayers, failed to extend neurites in response to nerve growth factor. Neither medium conditioned by endothelial cells nor fixed endothelial cells could by themselves induce all of these different phenomena in the PC12 cells. These results suggest that under coculture conditions PC12 cells change their state of differentiation toward a chromaffin cell-like phenotype. The rapid, transient increase in the expression of the protooncogene c-fos suggests that the mechanism(s) inducing the change in the state of differentiation in PC12 cells in coculture with the endothelial cells may be distinct from that described for the differentiation of PC12 cells--e.g., by glucocorticoids. We propose that similar interactions between endothelial cells and chromaffin cell precursors may occur during embryonic development and that these interactions might be instrumental for the organ-specific differentiation of the adrenal medulla in vivo.

  4. Effects of extremely low frequency magnetic fields on NGF induced neuronal differentiation of PC12 cells.

    PubMed

    Jung, In-Soo; Kim, Hyun-Jung; Noh, Ran; Kim, Soo-Chan; Kim, Chan-Wha

    2014-10-01

    Extremely low-frequency magnetic fields (ELF-MFs) affect various cellular processes and systems, such as cell proliferation, differentiation and metabolic pathways. The present study investigated ELF-MFs effect on nerve growth factor (NGF) induced neuronal differentiation of PC12 cells using proteomic applications to understand its role in the enhancement of neuronal differentiation. After 50 Hz, 1 mT ELF-MFs 5-day exposure on NGF induced PC12 cells, it was observed to increase neurite length as well as an increase in the number of neurite bearing cells. It was also discovered that there was a decrease in proliferation activity, which is associated with an increase in differentiated cells. Neuronal differentiation related mRNA levels and protein levels were increased in NGF induced PC12 cells. Compared with NGF induced group, ELF-MFs stimulated PC12 cells had different protein expression as measured with two-dimensional electrophoresis (2-DE) gels. Consequently six differentially expressed spots were detected between the 2-DE maps, which were identified by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF LC/MS/MS) as: peripherin, neurosecretory protein nerve growth factor inducible (VGF8a) precursor, dnaK-type molecular chaperone sp72-ps1 (HSP72-psI), low molecular weight (Mr) phosphotyrosine protein phosphatase isoenzyme AcP1 (LMW-PTP/ACP1), Tubulin alpha-1A (TUBA1A) chain, outcome predictor in acute leukemia 1 homolog (OPA1L). The identification of these proteins provides clues to the mechanism of ELF-MFs stimulation on NGF induced PC12 cells that occur during neuronal differentiation and may contribute to the development novel treatments for neurodegenerative diseases.

  5. DIFFERENTIAL MODULATION OF CATECHOLAMINES BY CHLOROTRIAZINE HERBICIDES IN PHEOCHROMOCYTOMA (PC12) CELLS IN VITRO

    EPA Science Inventory

    Differential modulation of catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro.

    Das PC, McElroy WK, Cooper RL.

    Curriculum in Toxicology, University of North Carolina, Chapel Hill 27599, USA.

    Epidemiological, wildlife, and lab...

  6. Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway.

    PubMed

    Wang, Xi; Lu, Xiaocheng; Zhu, Ronglan; Zhang, Kaixin; Li, Shuai; Chen, Zhongjun; Li, Lixin

    2017-01-25

    Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.

  7. Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells.

    PubMed Central

    Szeberényi, J; Cai, H; Cooper, G M

    1990-01-01

    A dominant inhibitory mutation of Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21 (Asn-17)Ha-ras] has been used to investigate the role of ras in neuronal differentiation of PC12 cells. The growth of PC12 cells, in contrast to NIH 3T3 cells, was not inhibited by p21(Asn-17)Ha-ras expression. However, PC12 cells expressing the mutant Ha-ras protein showed a marked inhibition of morphological differentiation induced by nerve growth factor (NGF) or fibroblast growth factor (FGF). These cells, however, were still able to respond with neurite outgrowth to dibutyryl cyclic AMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Induction of early-response genes (fos, jun, and zif268) by NGF and FGF but not by TPA was also inhibited by high levels of p21(Asn-17)Ha-ras. However, lower levels of p21(Asn-17) expression were sufficient to block neuronal differentiation without inhibiting induction of these early-response genes. Induction of the secondary-response genes SCG10 and transin by NGF, like morphological differentiation, was inhibited by low levels of p21(Asn-17) whether or not induction of early-response genes was blocked. Therefore, although inhibition of ras function can inhibit early-response gene induction, this is not required to block morphological differentiation or secondary-response gene expression. These results suggest that ras proteins are involved in at least two different pathways of signal transduction from the NGF receptor, which can be distinguished by differential sensitivity to p21(Asn-17)Ha-ras. In addition, ras and protein kinase C can apparently induce early-response gene expression by independent pathways in PC12 cells. Images PMID:2118994

  8. Nur77 is differentially modified in PC12 cells upon membrane depolarization and growth factor treatment.

    PubMed Central

    Hazel, T G; Misra, R; Davis, I J; Greenberg, M E; Lau, L F

    1991-01-01

    The rat pheochromocytoma cell line PC12 can be induced by growth factors to undergo proliferation and neuronal differentiation. These cells also have excitable membranes that can be depolarized by neurotransmitters or elevated levels of extracellular KCl. Treatment of PC12 cells with growth factors or membrane-depolarizing agents rapidly activates the expression of specific genes whose products are thought to mediate the subsequent biological responses. One such gene, nur77, is a member of the steroid and thyroid hormone receptor gene superfamily. We have identified the Nur77 protein and shown that it is synthesized rapidly and transiently in PC12 cells following stimulation, has a short half-life of 30 to 40 min, and is located in both the nucleus and the cytoplasm. Nur77 is posttranslationally modified, primarily by phosphorylation on serine residues. Phosphopeptide analysis reveals that Nur77 is modified differently upon membrane depolarization than after treatment with growth factors. We hypothesize that the activity of Nur77 is regulated by both differential gene expression and posttranslational modification and that these modes of regulation contribute to distinct downstream responses specific to membrane depolarization and growth factor treatment. Images PMID:1645447

  9. Poly(Dimethylsiloxane) (PDMS) Affects Gene Expression in PC12 Cells Differentiating into Neuronal-Like Cells

    PubMed Central

    Łopacińska, Joanna M.; Emnéus, Jenny; Dufva, Martin

    2013-01-01

    Introduction Microfluidics systems usually consist of materials like PMMA - poly(methyl methacrylate) and PDMS - poly(dimethylsiloxane) and not polystyrene (PS), which is usually used for cell culture. Cellular and molecular responses in cells grown on PS are well characterized due to decades of accumulated research. In contrast, the experience base is limited for materials used in microfludics chip fabrication. Methods The effect of different materials (PS, PMMA and perforated PMMA with a piece of PDMS underneath) on the growth and differentiation of PC12 (adrenal phaeochromocytoma) cells into neuronal-like cells was investigated using cell viability, cell cycle distribution, morphology, and gene expression analysis. Results/Conclusions After differentiation, the morphology, viability and cell cycle distribution of PC12 cells grown on PS, PMMA with and without PDMS underneath was the same. By contrast, 41 genes showed different expression for PC12 cells differentiating on PMMA as compared to on PS. In contrast, 677 genes showed different expression on PMMA with PDMS underneath as compared with PC12 cells on PS. The differentially expressed genes are involved in neuronal cell development and function. However, there were also many markers for neuronal cell development and functions that were expressed similarly in cells differentiating on PS, PMMA and PMMA with PDMS underneath. In conclusion, it was shown that PMMA has a minor impact and PDMS a major impact on gene expression in PC12 cells. PMID:23301028

  10. Differential susceptibility of naive and differentiated PC-12 cells to methylglyoxal-induced apoptosis: influence of cellular redox.

    PubMed

    Okouchi, Masahiro; Okayama, Naotsuka; Aw, Tak Yee

    2005-01-01

    Neuropathologies have been associated with neuronal de-differentiation and oxidative susceptibility. To address whether cellular states determines their oxidative vulnerability, we have challenged naive (undifferentiated) and nerve growth factor-induced differentiated pheochromocytoma (PC12) with methylglyoxal (MG), a model of carbonyl stress. MG dose-dependently induced greater apoptosis (24 h) in naive (nPC12) than differentiated (dPC12) cells. This enhanced nPC12 susceptibility was correlated with a high basal oxidized cellular glutathione-to-glutathione disulfide (GSH/GSSG) redox and an MG-induced GSH-to-Disulfide (GSSG plus protein-bound SSG) imbalance. The loss of redox balance occurred at 30 min post-MG exposure, and was prevented by N-acetylcysteine (NAC) that was unrelated to de novo GSH synthesis. NAC was ineffective when added at 1h post-MG, consistent with an early window of redox signaling. This redox shift was kinetically linked to decreased BcL-2, increased Bax, and release of mitochondrial cytochrome c which preceded caspase-9 and -3 activation and poly ADP-ribose polymerase (PARP) cleavage (1-2 h), consistent with mitochondrial apoptotic signaling. The blockade of apoptosis by cyclosporine A supported an involvement of the mitochondrial permeability transition pore. The enhanced vulnerability of nPC12 cells to MG and its relationship to cellular redox shifts will have important implications for understanding differential oxidative vulnerability in various cell types and their transition states.

  11. Chemogenomic analysis of neuronal differentiation with pathway changes in PC12 cells.

    PubMed

    Lin, Jack Yu-Shih; Wu, Chien Liang; Liao, Chia Nan; Higuchi, Akon; Ling, Qing-Dong

    2016-01-01

    The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database creates networks from interrelations between molecular biology and underlying chemical elements. This allows for analysis of biologic networks, genomic information, and higher-order functional information at a system level. Through high throughput experiments and system biology analysis, we investigated the genes and pathways associated with NGF induced neuronal differentiation. We performed microarray experiments and used the KEGG database, system biology analysis, and annotation of pathway functions to study NGF-induced differentiation in PC12 cells. We identified 2020 NGF-induced genes with altered expressions over time. Cross-matching with the KEGG database revealed 830 genes; among which, 395 altered genes were found to have a 2-fold increase in gene expression over a two-hour period. We then identified 191 associated biologic pathways in the KEGG database; the top 15 pathways showed correlation with neural differentiation. These included the neurotrophin pathways, mitogen-activated protein kinase (MAPK) pathways, genes associated with axonal guidance and the Wnt pathways. The activation of these pathways synchronized with nerve growth factor (NGF)-induced differentiation in PC12 cells. In summary, we have established a model system that allows one to systematically characterize the functional pathway changes in a group of neuronal population after an external stimulus.

  12. K(ATP) channel block prevents proteasome inhibitor-induced apoptosis in differentiated PC12 cells.

    PubMed

    Nam, Yoon Jeong; Lee, Da Hee; Lee, Min Sung; Lee, Chung Soo

    2015-10-05

    Dysfunction of the proteasome system has been suggested to be implicated in neuronal degeneration. Modulation of KATP channels appears to affect the viability of neuronal cells exposed to toxic insults. However, the effect of KATP channel blockers on the neuronal cell death mediated by proteasome inhibition has not been studied. The present study investigated the effect of KATP channel blockers on proteasome inhibitor-induced apoptosis in differentiated PC12 cells and SH-SY5Y cells. 5-Hydroxydecanoate (a selective KATP channel blocker) and glibenclamide (a cell surface and mitochondrial KATP channel inhibitor) reduced the proteasome inhibitor-induced apoptosis. Addition of the KATP channel blockers attenuated the proteasome inhibitor-induced changes in the levels of apoptosis-related proteins, the loss of the mitochondrial transmembrane potential, the increase in the formation of reactive oxygen species and the depletion of glutathione in both cell lines. The results show that KATP channel blockers may attenuate proteasome inhibitor-induced apoptosis in PC12 cells by suppressing activation of the mitochondrial pathway and of the caspase-8- and Bid-dependent pathways. The preventive effect appears to be associated with the inhibition of the formation of reactive oxygen species and the depletion of glutathione. KATP channel blockade appears to prevent proteasome inhibition-induced neuronal cell death.

  13. Green tea polyphenol epigallocatechin-3-gallate differentially modulates oxidative stress in PC12 cell compartments.

    PubMed

    Raza, Haider; John, Annie

    2005-09-15

    Tea polyphenols have been reported to be potent antioxidants and beneficial in oxidative stress related diseases. Prooxidant effects of tea polyphenols have also been reported in cell culture systems. In the present study, we have studied oxidative stress in the subcellular compartments of PC12 cells after treatment with different concentrations of the green tea polyphenol, epigallocatechin-3-gallate (EGCG). We have demonstrated that EGCG has differentially affected the production of reactive oxygen species (ROS), glutathione (GSH) metabolism and cytochrome P450 2E1 activity in the different subcellular compartments in PC12 cells. Our results have shown that although the cell survival was not inhibited by EGCG, there was, however, an increased DNA breakdown and activation of apoptotic markers, caspase 3 and poly- (ADP-ribose) polymerase (PARP) at higher concentrations of EGCG treatment. Our results suggest that the differential effects of EGCG might be related to the alterations in oxidative stress, GSH pools and CYP2E1 activity in different cellular compartments. These results may have implications in determining the chemopreventive therapeutic use of tea polyphenols in vivo.

  14. Boolean Modeling Reveals the Necessity of Transcriptional Regulation for Bistability in PC12 Cell Differentiation

    PubMed Central

    Offermann, Barbara; Knauer, Steffen; Singh, Amit; Fernández-Cachón, María L.; Klose, Martin; Kowar, Silke; Busch, Hauke; Boerries, Melanie

    2016-01-01

    The nerve growth factor NGF has been shown to cause cell fate decisions toward either differentiation or proliferation depending on the relative activity of downstream pERK, pAKT, or pJNK signaling. However, how these protein signals are translated into and fed back from transcriptional activity to complete cellular differentiation over a time span of hours to days is still an open question. Comparing the time-resolved transcriptome response of NGF- or EGF-stimulated PC12 cells over 24 h in combination with protein and phenotype data we inferred a dynamic Boolean model capturing the temporal sequence of protein signaling, transcriptional response and subsequent autocrine feedback. Network topology was optimized by fitting the model to time-resolved transcriptome data under MEK, PI3K, or JNK inhibition. The integrated model confirmed the parallel use of MAPK/ERK, PI3K/AKT, and JNK/JUN for PC12 cell differentiation. Redundancy of cell signaling is demonstrated from the inhibition of the different MAPK pathways. As suggested in silico and confirmed in vitro, differentiation was substantially suppressed under JNK inhibition, yet delayed only under MEK/ERK inhibition. Most importantly, we found that positive transcriptional feedback induces bistability in the cell fate switch. De novo gene expression was necessary to activate autocrine feedback that caused Urokinase-Type Plasminogen Activator (uPA) Receptor signaling to perpetuate the MAPK activity, finally resulting in the expression of late, differentiation related genes. Thus, the cellular decision toward differentiation depends on the establishment of a transcriptome-induced positive feedback between protein signaling and gene expression thereby constituting a robust control between proliferation and differentiation. PMID:27148350

  15. Boolean Modeling Reveals the Necessity of Transcriptional Regulation for Bistability in PC12 Cell Differentiation.

    PubMed

    Offermann, Barbara; Knauer, Steffen; Singh, Amit; Fernández-Cachón, María L; Klose, Martin; Kowar, Silke; Busch, Hauke; Boerries, Melanie

    2016-01-01

    The nerve growth factor NGF has been shown to cause cell fate decisions toward either differentiation or proliferation depending on the relative activity of downstream pERK, pAKT, or pJNK signaling. However, how these protein signals are translated into and fed back from transcriptional activity to complete cellular differentiation over a time span of hours to days is still an open question. Comparing the time-resolved transcriptome response of NGF- or EGF-stimulated PC12 cells over 24 h in combination with protein and phenotype data we inferred a dynamic Boolean model capturing the temporal sequence of protein signaling, transcriptional response and subsequent autocrine feedback. Network topology was optimized by fitting the model to time-resolved transcriptome data under MEK, PI3K, or JNK inhibition. The integrated model confirmed the parallel use of MAPK/ERK, PI3K/AKT, and JNK/JUN for PC12 cell differentiation. Redundancy of cell signaling is demonstrated from the inhibition of the different MAPK pathways. As suggested in silico and confirmed in vitro, differentiation was substantially suppressed under JNK inhibition, yet delayed only under MEK/ERK inhibition. Most importantly, we found that positive transcriptional feedback induces bistability in the cell fate switch. De novo gene expression was necessary to activate autocrine feedback that caused Urokinase-Type Plasminogen Activator (uPA) Receptor signaling to perpetuate the MAPK activity, finally resulting in the expression of late, differentiation related genes. Thus, the cellular decision toward differentiation depends on the establishment of a transcriptome-induced positive feedback between protein signaling and gene expression thereby constituting a robust control between proliferation and differentiation.

  16. The Ubiquitin Ligase Praja1 Reduces NRAGE Expression and Inhibits Neuronal Differentiation of PC12 Cells

    PubMed Central

    Teuber, Jan; Mueller, Bettina; Fukabori, Ryoji; Lang, Daniel; Albrecht, Anne; Stork, Oliver

    2013-01-01

    Evidence suggests that regulated ubiquitination of proteins plays a critical role in the development and plasticity of the central nervous system. We have previously identified the ubiquitin ligase Praja1 as a gene product induced during fear memory consolidation. However, the neuronal function of this enzyme still needs to be clarified. Here, we investigate its involvement in the nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma (PC12) cells. Praja1 co-localizes with cytoskeleton components and the neurotrophin receptor interacting MAGE homologue (NRAGE). We observed an enhanced expression of Praja1 after 3 days of NGF treatment and a suppression of neurite formation upon Praja1 overexpression in stably transfected PC12 cell lines, which was associated with a proteasome-dependent reduction of NRAGE levels. Our data suggest that Praja1, through ubiquitination and degradation of NRAGE, inhibits neuronal differentiation. The two murine isoforms, Praja1.1 and Praja1.2, appear to be functionally homologous in this respect. PMID:23717400

  17. NGF-conjugated iron oxide nanoparticles promote differentiation and outgrowth of PC12 cells

    NASA Astrophysics Data System (ADS)

    Marcus, M.; Skaat, H.; Alon, N.; Margel, S.; Shefi, O.

    2014-12-01

    The search for regenerative agents that promote neuronal differentiation and repair is of great importance. Nerve growth factor (NGF) which is an essential contributor to neuronal differentiation has shown high pharmacological potential for the treatment of central neurodegenerative diseases such as Alzheimer's and Parkinson's. However, growth factors undergo rapid degradation, leading to a short biological half-life. In our study, we describe a new nano-based approach to enhance the NGF activity resulting in promoted neuronal differentiation. We covalently conjugated NGF to iron oxide nanoparticles (NGF-NPs) and studied the effect of the novel complex on the differentiation of PC12 cells. We found that the NGF-NP treatment, at the same concentration as free NGF, significantly promoted neurite outgrowth and increased the complexity of the neuronal branching trees. Examination of neuronal differentiation gene markers demonstrated higher levels of expression in PC12 cells treated with the conjugated factor. By manipulating the NGF specific receptor, TrkA, we have demonstrated that NGF-NPs induce cell differentiation via the regular pathway. Importantly, we have shown that NGF-NPs undergo slower degradation than free NGF, extending their half-life and increasing NGF availability. Even a low concentration of conjugated NGF treatment has led to an effective response. We propose the use of the NGF-NP complex which has magnetic characteristics, also as a useful method to enhance NGF efficiency and activity, thus, paving the way for substantial neuronal repair therapeutics.The search for regenerative agents that promote neuronal differentiation and repair is of great importance. Nerve growth factor (NGF) which is an essential contributor to neuronal differentiation has shown high pharmacological potential for the treatment of central neurodegenerative diseases such as Alzheimer's and Parkinson's. However, growth factors undergo rapid degradation, leading to a short

  18. PC12 differentiation on biopolymer nanostructures

    NASA Astrophysics Data System (ADS)

    Cecchini, Marco; Bumma, Giorgia; Serresi, Michela; Beltram, Fabio

    2007-12-01

    The study of nervous system regeneration and axonal outgrowth control are relevant in several research areas, like neurophysiology or biomedical engineering. Among the elements that control neuron dynamics, the host substrate topography is a key parameter in determining cell differentiation. We present time-lapse experiments to analyze the differentiation dynamics of PC12 cells on nanopatterned biocompatible substrates. 200 nm depth gratings were fabricated on tissue-culture polystyrene substrates by nanoimprint lithography; different linewidths and pitches were compared down to 500 nm and 1000 nm, respectively. PC12 cells were cultured on these substrates and, following NGF administration to the medium, body morphology, cell movement and neuritogenesis were monitored at different time periods. In addition to demonstrating guided differentiation, our studies show complex time variations in body morphology and axon length, and guided cell movement. We show unstable synaptic connections and cell-body polarization, and the competition between topographical guidance and cell-cell interactions.

  19. Role for Egr1 in the Transcriptional Program Associated with Neuronal Differentiation of PC12 Cells

    PubMed Central

    Kletsov, Sergey; Lamm, Ryan J.; Elman, Jessica S.; Mullenbrock, Steven; Cooper, Geoffrey M.

    2017-01-01

    PC12 cells are a well-established model to study how differences in signal transduction duration can elicit distinct cell behaviors. Epidermal growth factor (EGF) activates transient ERK signaling in PC12 cells that lasts 30–60 min, which in turn promotes proliferation; nerve growth factor (NGF) activates more sustained ERK signaling that lasts 4–6 h, which in turns induces neuronal differentiation. Data presented here extend a previous study by Mullenbrock et al. (2011) that demonstrated that sustained ERK signaling in response to NGF induces preferential expression of a 69-member gene set compared to transient ERK signaling in response to EGF and that the transcription factors AP-1 and CREB play a major role in the preferential expression of several genes within the set. Here, we examined whether the Egr family of transcription factors also contributes to the preferential expression of the gene set in response to NGF. Our data demonstrate that NGF causes transient induction of all Egr family member transcripts, but a corresponding induction of protein was detected for only Egr1 and 2. Chromatin immunoprecipitation experiments provided clearest evidence that, after induction, Egr1 binds 12 of the 69 genes that are preferentially expressed during sustained ERK signaling. In addition, Egr1 expression and binding upstream of its target genes were both sustained in response to NGF versus EGF within the same timeframe that its targets are preferentially expressed. These data thus provide evidence that Egr1 contributes to the transcriptional program activated by sustained ERK signaling in response to NGF, specifically by contributing to the preferential expression of its target genes identified here. PMID:28076410

  20. Organic Photovoltaics and Bioelectrodes Providing Electrical Stimulation for PC12 Cell Differentiation and Neurite Outgrowth.

    PubMed

    Hsiao, Yu-Sheng; Liao, Yan-Hao; Chen, Huan-Lin; Chen, Peilin; Chen, Fang-Chung

    2016-04-13

    Current bioelectronic medicines for neurological therapies generally involve treatment with a bioelectronic system comprising a power supply unit and a bioelectrode device. Further integration of wireless and self-powered units is of practical importance for implantable bioelectronics. In this study, we developed biocompatible organic photovoltaics (OPVs) for serving as wireless electrical power supply units that can be operated under illumination with near-infrared (NIR) light, and organic bioelectronic interface (OBEI) electrode devices as neural stimulation electrodes. The OPV/OBEI integrated system is capable to provide electrical stimulation (ES) as a means of enhancing neuron-like PC12 cell differentiation and neurite outgrowth. For the OPV design, we prepared devices incorporating two photoactive material systems--β-carotene/N,N'-dioctyl-3,4,9,10-perylenedicarboximide (β-carotene/PTCDI-C8) and poly(3-hexylthiophene)/phenyl-C61-butyric acid methyl ester (P3HT/PCBM)--that exhibited open circuit voltages of 0.11 and 0.49 V, respectively, under NIR light LED (NLED) illumination. Then, we connected OBEI devices with different electrode gaps, incorporating biocompatible poly(hydroxymethylated-3,4-ethylenedioxythiophene), to OPVs to precisely tailor the direct current electric field conditions during the culturing of PC12 cells. This NIR light-driven OPV/OBEI system could be engineered to provide tunable control over the electric field (from 220 to 980 mV mm(-1)) to promote 64% enhancement in the neurite length, direct the neurite orientation on chips, or both. The OPV/OBEI integrated systems under NIR illumination appear to function as effective power delivery platforms that should meet the requirements for wirelessly offering medical ES to a portion of the nervous system; they might also be a key technology for the development of next-generation implantable bioelectronics.

  1. microRNA regulatory mechanism by which PLLA aligned nanofibers influence PC12 cell differentiation

    NASA Astrophysics Data System (ADS)

    Yu, Yadong; Lü, Xiaoying; Ding, Fei

    2015-08-01

    Objective. Aligned nanofibers (AFs) are regarded as promising biomaterials in nerve tissue engineering. However, a full understanding of the biocompatibility of AFs at the molecular level is still challenging. Therefore, the present study focused on identifying the microRNA (miRNA)-mediated regulatory mechanism by which poly-L-lactic acid (PLLA) AFs influence PC12 cell differentiation. Approach. Firstly, the effects of PLLA random nanofibers (RFs)/AFs and PLLA films (control) on the biological responses of PC12 cells that are associated with neuronal differentiation were examined. Then, SOLiD sequencing and cDNA microarray were employed to profile the expressions of miRNAs and mRNAs. The target genes of the misregulated miRNAs were predicted and compared with the mRNA profile data. Functions of the matched target genes (the intersection between the predicted target genes and the experimentally-determined, misregulated genes) were analyzed. Main results. The results revealed that neurites spread in various directions in control and RF groups. In the AF group, most neurites extended in parallel with each other. The glucose consumption and lactic acid production in the RF and AF groups were higher than those in the control group. Compared with the control group, 42 and 94 miRNAs were significantly dysregulated in the RF and AF groups, respectively. By comparing the predicted target genes with the mRNA profile data, five and 87 matched target genes were found in the RF and AF groups, respectively. Three of the matched target genes in the AF group were found to be associated with neuronal differentiation, whereas none had this association in the RF group. The PLLA AFs induced the dysregulation of miRNAs that regulate many biological functions, including axonal guidance, lipid metabolism and long-term potentiation. In particular, two miRNA-matched target gene-biological function modules associated with neuronal differentiation were identified as follows: (1) miR-23b, mi

  2. Triptolide Inhibited Cytotoxicity of Differentiated PC12 Cells Induced by Amyloid-Beta25–35 via the Autophagy Pathway

    PubMed Central

    Xu, Pengjuan; Li, Zhigui; Wang, Hui; Zhang, Xiaochen; Yang, Zhuo

    2015-01-01

    Evidence shows that an abnormal deposition of amyloid beta-peptide25–35 (Aβ25–35) was the primary cause of the pathogenesis of Alzheimer’s disease (AD). And the elimination of Aβ25–35 is considered an important target for the treatment of AD. Triptolide (TP), isolated from Tripterygium wilfordii Hook.f. (TWHF), has been shown to possess a broad spectrum of biological profiles, including neurotrophic and neuroprotective effects. In our study investigating the effect and potential mechanism of triptolide on cytotoxicity of differentiated rat pheochromocytoma cell line (the PC12 cell line is often used as a neuronal developmental model) induced by Amyloid-Beta25–35 (Aβ25–35), we used 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay, flow cytometry, Western blot, and acridine orange staining to detect whether triptolide could inhibit Aβ25–35–induced cell apoptosis. We focused on the potential role of the autophagy pathway in Aβ25–35-treated differentiated PC12 cells. Our experiments show that cell viability is significantly decreased, and the apoptosis increased in Aβ25–35-treated differentiated PC12 cells. Meanwhile, Aβ25–35 treatment increased the expression of microtubule-associated protein light chain 3 II (LC3 II), which indicates an activation of autophagy. However, triptolide could protect differentiated PC12 cells against Aβ25–35-induced cytotoxicity and attenuate Aβ25–35-induced differentiated PC12 cell apoptosis. Triptolide could also suppress the level of autophagy. In order to assess the effect of autophagy on the protective effects of triptolide in differentiated PC12 cells treated with Aβ25–35, we used 3-Methyladenine (3-MA, an autophagy inhibitor) and rapamycin (an autophagy activator). MTT assay showed that 3-MA elevated cell viability compared with the Aβ25–35-treated group and rapamycin inhibits the protection of triptolide. These results suggest that triptolide will repair the

  3. Neuronal differentiation of PC12 and embryonic stem cells in two- and three-dimensional in vitro culture.

    PubMed

    Sadri, Soheil; Khazaei, Mozafar; Ghanbari, Ali; Khazaei, Mohammad Rasool; Shah, Palak

    2014-04-01

    The quality of neuronal differentiation and reduction in apoptosis that occurred in two-dimensional (2D) and three-dimensional (3D) culture conditions is compared. PC12 and embryonic stem cells are two commonly utilized cell lines for the study of neuronal regeneration. These cells were induced to neuronally differentiate by adding NGF and retinoic acid respectively. Total neurite length and expression of neuronal markers (MAP-2 and beta-tubulin) was assessed by morphometry and immunocytochemistry. Also, TUNEL assay was used to detect apoptosis. Upon exposure to a differentiation media in the 3D fibrin gel, PC12 and embryonic stem cells stopped dividing, had increased adhesion to the substratum, extended neurite processes and expressed neuronal markers. The same results, however, were not observedwith the 2D culture. Also, the apoptosis index performed by TUNEL a ss ay demonstrated a reduction in th e degree of apoptosis in the 3D culture compared to 2D culture. Fibrin matrix supports growth and n euronal differentiation of PC12 andembryonic stem cells. In addition, the 3D culture enhanced cellular resistance to apoptosis when compared to the 2D culture. It appears as if a 3D culture system may offer a better technique for future neuronal tissue engineering investigations.

  4. NGF induction of the gene encoding the protease transin accompanies neuronal differentiation in PC12 cells.

    PubMed

    Machida, C M; Rodland, K D; Matrisian, L; Magun, B E; Ciment, G

    1989-06-01

    Various proteases have been found to be released by the growth cones of developing neurons in culture and have been hypothesized to play a role in the process of axon elongation. We report here that nerve growth factor (NGF) induced the gene encoding the metalloprotease transin in PC12 cells with a time course coincident with the initial appearance of neurites by these cells. Acidic and basic fibroblast growth factors also stimulated transin mRNA expression and neurite outgrowth, whereas various other agents had no effects on either of these phenomena. In contrast, dexamethasone was found to inhibit the induction of transin mRNA when added with, or following, NGF treatment. Finally, we show that sequences contained within 750 bp of the 5' untranscribed region of the transin gene confer responsiveness to NGF and dexamethasone.

  5. [NEURONAL DIFFERENTIATION OF PC12 CELL LINE AND MURINE NEURAL STEM CELLS ON THE CARBON NANOTUBES FILMS].

    PubMed

    Posypanova, G A; Gaiduchenko, A I; Moskaleva, E Yu; Fedorov, G E

    2016-01-01

    The study of the interaction of nerve cells with specially designed substrates (scaffolds) with different surface characteristics at the nanoscale is a necessary step in the development of methods of stimulation of regeneration of nervous tissues, as well as to create next generation of bioelectronic devices. A promising material for such scaffolds may be carbon nanotubes (CNT) that are flexible films of graphene rolled into nano-sized cylindrical tubes. CNT were produced by chemical deposition from the gas phase. The analysis of the PC12 cells cultivated on quartz glass coated by carbon nanotubes films using electron and light microscopy has shown that CNT stimulate the proliferation and do not inhibit neuronal differentiation of PC12 cells. We have found that it is possible to obtain differentiated neurons from murine neural stem cells on the quartz glasses covered with CNT films. The data obtained indicate that the CNT films produced by chemical deposition from the gas phase onto quartz glass may be used as the electro conductive scaffold to obtain and study the functions of neural cells and possibly of mature neurons.

  6. Flavanonol taxifolin attenuates proteasome inhibition-induced apoptosis in differentiated PC12 cells by suppressing cell death process.

    PubMed

    Nam, Yoon Jeong; Lee, Da Hee; Shin, Yong Kyoo; Sohn, Dong Suep; Lee, Chung Soo

    2015-03-01

    The proteasomal dysfunction and mitochondrial impairment has been implicated in neuronal degeneration. Taxifolin has antioxidant and anti-inflammatory effects. However, the effect of taxifolin on the neuronal cell death induced by proteasome inhibition has not been studied. Therefore, in the respect of cell death process, we assessed the effect of taxifolin on the proteasome inhibition-induced apoptosis in neuronal cell injury using differentiated PC12 cells. The proteasome inhibitors MG132 and MG115 induced a decrease in Bid, Bcl-2, and survivin protein levels, an increase in Bax, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases(-8, -9 and -3), an increase in the tumor suppressor p53 levels and cleavage of PARP-1. The addition of taxifolin attenuated the proteasome inhibitor-induced changes in the apoptosis-related protein levels, formation of reactive oxygen species, depletion and oxidation of GSH, formations of malondialdehyde and carbonyls, and cell death. The results show that taxifolin may attenuate the proteasome inhibitor-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The preventive effect of taxifolin appears to be attributed to its inhibitory effect on the formation of reactive oxygen species, and depletion and oxidation of GSH.

  7. Differential protection of pre-, co- and post-treatment of curcumin against hydrogen peroxide in PC12 cells.

    PubMed

    Siddiqui, M A; Kashyap, M P; Kumar, V; Tripathi, V K; Khanna, V K; Yadav, S; Pant, A B

    2011-03-01

    Pharmacological potential of curcumin was assessed in PC12 cells against hydrogen peroxide (H(2) O(2)) exposure. In MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays, 24-hour exposure of H(2)O(2) (0.5 mM and above) was found to be cytotoxic. A significant (p < 0.001) increase in percentage cell viability was recorded in PC12 cells pretreated with curcumin (25, 50 and 100 µg/mL) for 24 hours prior to H(2)O(2) (0.5 and 1 mM) exposure for 24 hours. Co-exposure to H(2)O(2) and curcumin was also found effective. However, a therapeutic treatment of curcumin for 24 hours after H(2)O(2) exposure to the cells was found ineffective. Differential response of PC12-H(2)O(2) model to curcumin in MTT and LDH assays suggests the utility of these endpoints to sort the drug candidates to study their antioxidant potential.

  8. The induction of tuftelin expression in PC12 cell line during hypoxia and NGF-induced differentiation.

    PubMed

    Leiser, Yoav; Silverstein, Nechama; Blumenfeld, Anat; Shilo, Dekel; Haze, Amir; Rosenfeld, Eli; Shay, Boaz; Tabakman, Rinat; Lecht, Shimon; Lazarovici, Philip; Deutsch, Dan

    2011-01-01

    The tuftelin protein isoforms undergo post-translation modifications, and are ubiquitously expressed in various tissues in embryos, adults, and tumors. Developmental and pathological studies suggested an apparent correlation between oxygen deprivation and tuftelin expression. The aim of the study was therefore to investigate the effect of a pathological insult (hypoxia) and a physiological growth factor (NGF), which antagonistically regulate HIF1 expression, on tuftelin expression using the neuronal PC12 cell model. In the present study, we first demonstrated the expression of tuftelin in PC12 cells, providing an experimental system to investigate the pathophysiological role of tuftelin. Furthermore, we demonstrated the induction of tuftelin during hypoxia by oxygen deprivation and during chemical hypoxia by cobalt chloride. Down-regulation of HIF1α mRNA blocked hypoxia-induced HIF1α expression, and reduced by 89% hypoxia-induced tuftelin expression. In mice, intraperitoneal injection of cobalt chloride significantly induced tuftelin mRNA and protein expression in the brain. During NGF-mediated PC12 differentiation, tuftelin expression was significantly induced in correlation with neurite outgrowth. This induction was partially blocked by K252a, a selective antagonist of the NGF receptor TrkA, indicating the involvement of the TrkA-signaling pathways in tuftelin induction by NGF. Revealing the physiological role of tuftelin will clarify mechanisms related to the "hypoxic genome," and NGF-induced neurotrophic and angiogenic effects.

  9. The deubiquitinating enzyme UBPy/USP8 interacts with TrkA and inhibits neuronal differentiation in PC12 cells.

    PubMed

    Ceriani, Michela; Amigoni, Loredana; D'Aloia, Alessia; Berruti, Giovanna; Martegani, Enzo

    2015-04-10

    The tropomyosin-related kinase (Trk) family of receptor tyrosine kinases controls synaptic function, plasticity and sustains differentiation, morphology, and neuronal cell survival. Understanding Trk receptors down-regulation and recycling is a crucial step to point out sympathetic and sensory neuron function and survival. PC12 cells derived from pheochromocytoma of the rat adrenal medulla have been widely used as a model system for studies of neuronal differentiation as they respond to nerve growth factor (NGF) with a dramatic change in phenotype and acquire a number of properties characteristic of sympathetic neurons. In this study we demonstrated that in PC12 cells the TrkA receptor interacts with the deubiquitinating enzyme USP8/UBPy in a NGF-dependent manner and that it is deubiquitinated in vivo and in vitro by USP8. USP8 overexpression blocked NGF-induced neurites outgrowth while the overexpression of the catalytically inactive mutant USP8/UBPy(C748A) caused a marked increase of cell differentiation. Localization and biochemical experiments have point out that USP8 and TrkA partially co-localize in endosomes after NGF stimulation. Finally we have studied the role played by USP8 on TrkA turnover; using specific siRNA for USP8 we found that USP8 knockdown increases TrkA half-life, suggesting that the deubiquitinating activity of USP8 promotes TrkA degradation.

  10. MECHANISMS OF MANGANESE-INDUCED RAT PHEOCHROMOCYTOMA (PC12) CELL DEATH AND CELL DIFFERENTIATION. (R826248)

    EPA Science Inventory

    Mn is a neurotoxin that leads to a syndrome resembling Parkinson's disease after prolonged exposure to high concentrations. Our laboratory has been investigating the mechanism by which Mn induces neuronal cell death. To accomplish this, we have utilized rat pheochromocytom...

  11. Concomitant inhibition of prolyl hydroxylases and ROCK initiates differentiation of mesenchymal stem cells and PC12 towards the neuronal lineage.

    PubMed

    Pacary, Emilie; Petit, Edwige; Bernaudin, Myriam

    2008-12-12

    This study demonstrates that a prolyl hydroxylase inhibitor, FG-0041, is able, in combination with the ROCK inhibitor, Y-27632, to initiate differentiation of mesenchymal stem cells (MSCs) into neuron-like cells. FG-0041/Y-27632 co-treatment provokes morphological changes into neuron-like cells, increases neuronal marker expression and provokes modifications of cell cycle-related gene expression consistent with a cell cycle arrest of MSC, three events showing the engagement of MSC towards the neuronal lineage. Moreover, as we observed in our previous studies with cobalt chloride and desferroxamine, the activation of HIF-1 by this prolyl hydroxylase inhibitor is potentiated by Y-27632 which could explain at least in part the effect of this co-treatment on MSC neuronal differentiation. In addition, we show that this co-treatment enhances neurite outgrowth and tyrosine hydroxylase expression in PC12 cells. Altogether, these results evidence that concomitant inhibition of prolyl hydroxylases and ROCK represents a relevant protocol to initiate neuronal differentiation.

  12. The effect of Cyclin-dependent kinase 5 on voltage-dependent calcium channels in PC12 cells varies according to channel type and cell differentiation state.

    PubMed

    Furusawa, Kotaro; Asada, Akiko; Saito, Taro; Hisanaga, Shin-ichi

    2014-08-01

    Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase that plays an important role in the release of neurotransmitter from pre-synaptic terminals triggered by Ca(2+) influx into the pre-synaptic cytoplasm through voltage-dependent Ca(2+) channels (VDCCs). It is reported that Cdk5 regulates L-, P/Q-, or N-type VDCC, but there is conflicting data as to the effect of Cdk5 on VDCC activity. To clarify the mechanisms involved, we examined the role of Cdk5 in regulating the Ca(2+) -channel property of VDCCs, using PC12 cells expressing endogenous, functional L-, P/Q-, and N-type VDCCs. The Ca(2+) influx, induced by membrane depolarization with high K(+) , was monitored with a fluorescent Ca(2+) indicator protein in both undifferentiated and nerve growth factor (NGF)-differentiated PC12 cells. Overall, Ca(2+) influx was increased by expression of Cdk5-p35 in undifferentiated PC12 cells but suppressed in differentiated PC12 cells. Moreover, we found that different VDCCs are distinctly regulated by Cdk5-p35 depending on the differentiation states of PC12 cells. These results indicate that Cdk5-p35 regulates L-, P/Q-, or N-type VDCCs in a cellular context-dependent manner. Calcium (Ca(2+) ) influx through voltage-dependent Ca(2+) channels (VDCCs) triggers neurotransmitter release from pre-synaptic terminal of neurons. The channel activity of VDCCs is regulated by Cdk5-p35, a neuronal Ser/Thr kinase. However, there have been debates about the regulation of VDCCs by Cdk5. Using PC12 cells, we show that Cdk5-p35 regulates VDCCs in a type (L, P/Q, and N) and differentiation-dependent manner. NGF = nerve growth factor.

  13. EVALUATION OF PROTEIN MARKERS FOR NEURONAL DIFFERENTIATION IN PC12 CELLS.

    EPA Science Inventory

    Chemical-induced injury of the developing nervous system can be manifested as a change in the differentiation or growth of neurons. The present study evaluated the use of proteins associated with axonal growth and synaptogenesis as markers for neuronal differentiation in vitro. ...

  14. SUB-ACUTE TREATMENT WITH METHYLMERCURY DURING DIFFERENTIATION OF PHEOCHROMOCYTOMA (PC12) CELLS DOES NOT ALTER BINDING OF ION CHANNEL LIGANDS OR CELL MORPHOLOGY.

    EPA Science Inventory

    We demonstrated recently that 6 days of exposure to nanomolar concentrations (3-10 nM) of methylmercury (MeHg) during nerve growth factor (NGF) induced PC12 cell differentiation reduced the amplitude and density of voltage-gated sodium and calcium currents. In the present study,...

  15. (-)-3,5-Dicaffeoyl-muco-quinic acid isolated from Aster scaber contributes to the differentiation of PC12 cells: through tyrosine kinase cascade signaling.

    PubMed

    Hur, Jin Young; Lee, Pyeongjae; Kim, Hocheol; Kang, Insug; Lee, Kang Ro; Kim, Sun Yeou

    2004-01-23

    Aster scaber T. (Asteraceae) has been used in traditional Korean and Chinese medicine to treat bruises, snakebites, headaches, and dizziness. (-)-3,5-Dicaffeoyl-muco-quinic acid (DQ) isolated from A. scaber induced neurite outgrowth in PC12 cells. It has been reported that the activation of the extracellular signal regulated kinase 1/2 (Erk 1/2) and phosphoinositide 3 (PI3) kinase plays a crucial role in the NGF-induced differentiation of PC12 cells. This study showed that the effect of DQ on neurite outgrowth is mediated via the Erk 1/2 and PI3 kinase-dependent pathways like NGF. Furthermore, DQ stimulated the phosphorylation of Trk A. Overall, DQ elicited the differentiation of PC12 cells through Trk A phosphorylation followed by Erk 1/2 and PI3 kinase activation.

  16. Maslinic Acid Protected PC12 Cells Differentiated by Nerve Growth Factor against β-Amyloid-Induced Apoptosis.

    PubMed

    Yang, Yu-wan; Tsai, Chia-wen; Mong, Mei-chin; Yin, Mei-chin

    2015-12-02

    β-Amyloid peptide (Abeta) was used to induce apoptosis in PC12 cells differentiated by nerve growth factor, and the protective activities of maslinic acid (MA) at 2-16 μM were examined. Abeta treatment lowered Bcl-2 expression, raised Bax expression, and decreased cell viability. MA pretreatments decreased Bax expression, raised the Bcl-2/Bax ratio, and increased cell viability. MA pretreatments retained glutathione content and decreased subsequent Abeta-induced release of reactive oxygen species, tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. Abeta treatment up-regulated protein expression of p47(phox), gp91(phox), mitogen-activated protein kinase, advanced glycation end product receptor (RAGE), and nuclear factor-κ B (NF-κB). MA pretreatments at 2-16 μM suppressed the expression of proteins including gp91(phox), p47(phox), p-p38, and NF-κB p65, at 4-16 μM down-regulated RAGE and NF-κB p50 expression, and at 8 and 16 μM reduced p-ERK1/2 expression. These novel findings suggest that maslinic acid is a potent compound against Abeta-induced cytotoxicity.

  17. Inhibitory effects of multiwall carbon nanotubes with high iron impurity on viability and neuronal differentiation in cultured PC12 cells.

    PubMed

    Meng, Li; Jiang, Aihua; Chen, Rui; Li, Chen-zhong; Wang, Liming; Qu, Ying; Wang, Peng; Zhao, Yuliang; Chen, Chunying

    2013-11-08

    The increasing use of carbon nanotubes (CNTs) in biomedical applications has garnered a great concern on their potential negative effects to human health. CNTs have been reported to potentially disrupt normal neuronal function and they were speculated to accumulate and cause brain damage, although a lot of distinct and exceptional properties and potential wide applications have been associated with this material in neurobiology. Fe impurities strapped inside the CNTs may be partially responsible for neurotoxicity generation. In the present study, we selected rat pheochromocytoma (PC12) cells to investigate and compare the effects of two kinds of multiwall carbon nanotubes (MWCNTs) with different concentrations of Fe impurities which usually come from the massive production of CNTs by chemical vapor deposition. Exposure to Fe-high MWCNTs can reduce cell viability and increase cytoskeletal disruption of undifferentiated PC12 cells, diminish the ability to form mature neurites, and then adversely influence the neuronal dopaminergic phenotype in NGF-treated PC-12 cells. The present results highlight the critical role of iron residue in the adverse response to MWCNTs exposure in neural cells. These findings provide useful information for understanding the toxicity and safe application of carbon nanotubes.

  18. Exposure of PC12 cells to NGF/ethanol results in accelerated differentiation and altered gene expression

    SciTech Connect

    White, K.R.; Wooten, M.W. )

    1991-03-11

    The role of alcohols in affecting neuromorphogenesis was investigated in the pheochromocytoma cell line, PC12. The effect of ethanol at physiological concentrations in this system leads to accelerated neurite extension in the presence of suboptimal concentrations of NGF. Accelerated morphological differentiation was dependent upon the side chain length of the alcohol and not inhibited by pyrazole. Ethanol/NGF induced neurite extension can be blocked with 50nM of K252a, but not sphingozine, H7, H89, genistein or okadaic acid. Changes in the expression of 17 NGF-induced and/or neuronal transcripts were examined in relationship to time of NGF/ethanol exposure; dose of NGF/ethanol; and side-chain length of NFG/alcohol. The authors studies indicate that ethanol potentiates the effects of NFG and subsequent neurogenesis through both protein kinase C and cAMP-independent pathways. In addition, these data show that ethanol is capable of altering gene expression in a specific manner.

  19. Over-expression of the Sirt3 sirtuin Protects neuronally differentiated PC12 Cells from degeneration induced by oxidative stress and trophic withdrawal.

    PubMed

    Shulyakova, Natalya; Sidorova-Darmos, Elena; Fong, Jamie; Zhang, Guangming; Mills, Linda R; Eubanks, James H

    2014-10-31

    Sirt3 is a mitochondrial sirtuin whose deacetylase activity regulates facets of oxidative metabolic efficiency, anti-oxidative capacity, and intra-mitochondrial signaling. In this study, we tested whether the over-expression of a human Sirt3-myc transgene in differentiated PC12 cells, a model of sympathetic catecholaminergic neurons, would affect the sensitivity of these cells to oxidative stress or trophic withdrawal insults. Expression analysis revealed the Sirt3-myc product was expressed as a 45kDa pro-form, which localized primarily within the cytosol, and a 30kDa processed form that localized predominantly within mitochondria. When subjected to acute glucose deprivation or acute oxygen-glucose deprivation, differentiated PC12 cells over-expressing Sirt3-myc displayed significantly lower levels of cytotoxicity, both at the end of the insult, and at different times following media reperfusion, than cells transfected with a control plasmid. Further, Sirt3-myc over-expression also protected differentiated PC12 cells from apoptosis induced by trophic withdrawal. Collectively, these data indicate that an elevation of Sirt3 is sufficient to protect neuronal PC12 cells from cytotoxic insults, and add to the growing evidence that Sirt3 could be targeted for neuroprotective intervention.

  20. Inhibitory effects of psychotomimetic sigma ligands on nicotine-induced K+ flux from differentiated PC12 cells.

    PubMed

    Yamamoto, H; Sagi, N; Yamamoto, T; Goji, Y; Okuwa, M; Yoshii, M; Moroji, T

    1992-11-23

    In NGF-treated PC12 cells, nicotine-induced K+ release was measured with a K(+)-sensitive microelectrode. The K+ outflow via nicotinic ACh receptor cation channels was inhibited by various psychotomimetic sigma ligands in the sequence of PCP, dextromethorphan > DTG, MK 801, (+)SKF10047 > (+)3-PPP. The K+ release was not affected by the neuroleptic sigma ligand haloperidol nor by the calcium antagonist nifedipine. The results suggest that psychotomimetic sigma ligands inhibit nicotine-stimulated K+ flux by interacting with nicotinic, rather than via sigma 2 receptors.

  1. Neuroprotective effects of nimodipine and nifedipine in the NGF-differentiated PC12 cells exposed to oxygen-glucose deprivation or trophic withdrawal.

    PubMed

    Lecht, Shimon; Rotfeld, Elena; Arien-Zakay, Hadar; Tabakman, Rinat; Matzner, Henry; Yaka, Rami; Lelkes, Peter I; Lazarovici, Philip

    2012-10-01

    The goal of this study was to compare the neuroprotective properties of the L-type Ca²⁺ channel blockers, nimodipine and nifedipine, using nerve growth factor (NGF)-differentiated PC12 neuronal cultures exposed to oxygen-glucose deprivation (OGD) and trophic withdrawal-induced cell death. Nimodipine (1-100 μM) conferred 65±13% neuroprotection upon exposure to OGD and 35±6% neuroprotection towards different trophic withdrawal-induced cell death measured by lactate dehydrogenase and caspase 3 activities. The time window of nimodipine conferred neuroprotection was detected during the first 5h but not at longer OGD exposures. Nifedipine (1-100 μM), to a lower potency than nimodipine, conferred 30-55±8% neuroprotection towards OGD in PC12 cells and 29±5% in rat hypocampal slices, and 10±3% neuroprotection at 100 μM towards trophic withdrawal-induced PC12 cell death. The ability to demonstrate that nimodipine conferred neuroprotection in a narrow therapeutic time-window indicates that the OGD PC12 model mimics the in vivo models and therefore suitable for neuroprotective drug discovery and development.

  2. RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells.

    PubMed

    Tam, See-Ying; Lilla, Jennifer N; Chen, Ching-Cheng; Kalesnikoff, Janet; Tsai, Mindy

    2015-01-01

    Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.

  3. Dynamic change of neural cell adhesion molecule polysialylation on human neuroblastoma (IMR-32) and rat pheochromocytoma (PC-12) cells during growth and differentiation.

    PubMed

    Poongodi, Geetha L; Suresh, Nimmagadda; Gopinath, Subash C B; Chang, Tschining; Inoue, Sadako; Inoue, Yasuo

    2002-08-02

    Polysialic acid (PSA) is a regulatory epitope of neural cell adhesion molecule (NCAM) in homophilic adhesion of neural cells mediated by NCAM, is also known to be re-expressed in several human tumors, thus serves as an oncodevelopmental antigen. In this study, using a recently developed ultrasensitive chemical method in addition to immunochemical methods, growth stage-dependent and retinoic acid (RA)-induced differentiation-dependent changes of PSA expression in human neuroblastoma (IMR-32) and rat pheochromocytoma (PC-12) cells were analyzed both qualitatively and quantitatively. Both IMR-32 and PC-12 cells expressed PSA on NCAM, and the level of PSA expressed per unit weight of cells increased with post-inoculation incubation time. The most prominent feature was seen at the full confluence stage. RA induced neuronal differentiation in both IMR-32 and CP-12 cells that paralleled the change in the PSA level. Chemical analysis revealed the presence of NCAM glycoforms differing in the degree of polymerization (DP) of oligo/polysialyl chains, whose DP was smaller than 40. DP distribution of PSA was different between the cell lines and was changed by the growth stage and the RA treatment. Thus DP analysis of PSA is important in understanding both mechanism and biological significance of its regulated expression.

  4. Asarone from Acori Tatarinowii Rhizoma Potentiates the Nerve Growth Factor-Induced Neuronal Differentiation in Cultured PC12 Cells: A Signaling Mediated by Protein Kinase A

    PubMed Central

    Lam, Kelly Y. C.; Chen, Jianping; Lam, Candy T. W.; Wu, Qiyun; Yao, Ping; Dong, Tina T. X.; Lin, Huangquan; Tsim, Karl W. K.

    2016-01-01

    Acori Tatarinowii Rhizoma (ATR), the rhizome of Acorus tatarinowii Schott, is being used clinically to treat neurological disorders. The volatile oil of ATR is being considered as an active ingredient. Here, α-asarone and β-asarone, accounting about 95% of ATR oil, were evaluated for its function in stimulating neurogenesis. In cultured PC12 cells, application of ATR volatile oil, α-asarone or β-asarone, stimulated the expression of neurofilaments, a bio-marker for neurite outgrowth, in a concentration-dependent manner. The co-treatment of ATR volatile oil, α-asarone or β-asarone, with low concentration of nerve growth factor (NGF) potentiated the NGF-induced neuronal differentiation in cultured PC12 cells. In addition, application of protein kinase A inhibitors, H89 and KT5720, in cultures blocked the ATR-induced neurofilament expression, as well as the phosphorylation of cAMP-responsive element binding protein (CREB). In the potentiation of NGF-induced signaling in cultured PC12 cells, α-asarone and β-asarone showed synergistic effects. These results proposed the neurite-promoting asarone, or ATR volatile oil, could be useful in finding potential drugs for treating various neurodegenerative diseases, in which neurotrophin deficiency is normally involved. PMID:27685847

  5. Apocynin attenuates cholesterol oxidation product-induced programmed cell death by suppressing NF-κB-mediated cell death process in differentiated PC12 cells.

    PubMed

    Lee, Da Hee; Nam, Yoon Jeong; Lee, Chung Soo

    2015-10-01

    Cholesterol oxidation products are suggested to be involved in neuronal degeneration. Apocynin has demonstrated to have anti-inflammatory and anti-oxidant effects. We assessed the effect of apocynin on the cholesterol oxidation product-induced programmed cell death in neuronal cells using differentiated PC12 cells in relation to NF-κB-mediated cell death process. 7-Ketocholesterol and 25-hydroxycholesterol decreased the levels of Bid and Bcl-2, increased the levels of Bax and p53, and induced loss of the mitochondrial transmembrane potential, release of cytochrome c and activation of caspases (-8, -9 and -3). 7-Ketocholesterol caused an increase in the levels of cytosolic and nuclear NF-κB p65, cytosolic NF-κB p50 and cytosolic phospho-IκB-α, which was inhibited by the addition of 0.5 μM Bay11-7085 (an inhibitor of NF-κB activation). Apocynin attenuated the cholesterol oxidation product-induced changes in the programmed cell death-related protein levels, NF-κB activation, production of reactive oxygen species, and depletion of GSH. The results show that apocynin appears to attenuate the cholesterol oxidation product-induced programmed cell death in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways that are mediated by NF-κB activation. The preventive effect appears to be associated with the inhibitory effect on the production of reactive oxygen species and depletion of GSH.

  6. A "classical" homodimeric erythropoietin receptor is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells.

    PubMed

    Um, Moonkyoung; Gross, Alec W; Lodish, Harvey F

    2007-03-01

    The hematopoietic cytokine erythropoietin (Epo) exerts cytoprotective effects on several types of neuronal cells both in vivo and in culture. Detailed molecular mechanisms underlying this phenomenon have not been elucidated and even the identity of the cytoprotective Epo receptors in neuronal cells is controversial. Here we show that Epo prevents staurosporine-induced apoptosis of differentiated human neuroblastoma SH-SY5Y cells, and activates the STAT5, AKT and MAPK signaling pathways. Differentiated SH-SY5Y cells have fewer than 50 high affinity Epo surface binding sites per cell, which could not be detected by standard assays measuring binding of 125I-labeled Epo. However, by measuring endocytosis of 125I-Epo, we could reliably quantify very small numbers of high-affinity Epo surface binding sites. Using SH-SY5Y cells stably expressing an Epo receptor (EpoR) shRNA and thus lacking detectable EpoR expression, we show that high affinity binding of Epo to these neuronal cells is mediated by the hematopoietic EpoR, and that this EpoR is also essential for the antiapoptotic activity of Epo. In contrast, a mutant Epo that has an intact binding site 1 but a non-functional binding site 2 and hence binds only to one cell surface EpoR molecule ("site 2" Epo mutant) displays significantly lower antiapoptotic activity than wild-type Epo. Furthermore, expression of the GM-CSF/IL-3/IL-5 receptor common beta chain, which was proposed to be responsible for the cytoprotective activity of Epo on certain types of neuronal cells, was undetectable in differentiated SH-SY5Y cells. Epo also alleviated staurosporine-induced apoptosis of rat PC-12 pheochromocytoma cells while the R103A "site 2" Epo mutant did not, and we could not detect expression of the common beta chain in PC-12 cells. Together our results indicate that Epo exerts its antiapoptotic effects on differentiated SH-SY5Y and PC-12 cells through the standard stoichiometry of one molecule of Epo binding to two EpoR subunits

  7. Anisomycin uses multiple mechanisms to stimulate mitogen-activated protein kinases and gene expression and to inhibit neuronal differentiation in PC12 phaeochromocytoma cells.

    PubMed

    Törocsik, B; Szeberényi, J

    2000-02-01

    Treatment of PC12 cells with nerve growth factor (NGF) stimulates extracellular signal-regulated kinases (ERKs), as well as stress-activated c-Jun N-terminal kinases (JNKs) and p38 kinase, and induces neuronal differentiation. While the pivotal role of ERKs in NGF-induced morphological differentiation is well established, the contribution of JNK- and p38-pathways is less clear. The role of the JNK- and p38-pathway in PC12 cells was analysed by using anisomycin, a protein synthesis inhibitor that activates JNKs and p38. Non-toxic concentrations of anisomycin were found to stimulate these enzyme activities as well as the expression of the early response genes c-jun, c-fos and zif268, and to inhibit NGF-induced neurite formation. These effects of anisomycin appear to be mediated by the generation of reactive oxygen species (ROS), which in turn act through both TrkA/Ras-dependent and -independent signalling pathways. In addition, cross-talk between the p38- and ERK-pathways appears to play a role in the action of anisomycin.

  8. Microarray and synchronization of neuronal differentiation with pathway changes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) databank in nerve growth factor-treated PC12 cells.

    PubMed

    Lin, Chih-Ming; Feng, Wayne

    2012-08-01

    The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database creates networks from interrelations between molecular biology and underlying chemical elements. This allows for analysis of biologic networks, genomic information, and higher-order functional information at a systems level. We performed microarray experiments and used the KEGG database, systems biology analysis, and annotation of pathway function to study nerve growth factor (NGF)-induced differentiation of PC12 cells. Cells were cultured to 70%-80% confluence, treated with NGF for 1 or 3 hours (h), and RNA was extracted. Stage 1 data analysis involved analysis of variance (ANOVA), and stage 2 involved cluster analysis and heat map generation. We identified 2020 NGF-induced PC12 genes (1038 at 1 h and 1554 at 3 h). Results showed changes in gene expression over time. We compared these genes with 6035 genes from the KEGG database. Cross-matching resulted in 830 genes. Among these, we identified 395 altered genes (155 at 1 h and 301 at 3 h; 2-fold increase from 1 h to 3 h). We identified 191 biologic pathways in the KEGG database; the top 15 showed correlations with neuronal differentiation (mitogen-activated protein kinase [MAPK] pathway: 35 genes at 1 h, 54 genes at 3 h; genes associated with axonal guidance: 12 at 1 h, 26 at 3 h; Wnt pathway: 16 at 1 h, 25 at 3 h; neurotrophin pathway: 4 at 1 h, 14 at 3 h). Thus, we identified changes in neuronal differentiation pathways with the KEGG database, which were synchronized with NGF-induced differentiation.

  9. Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth.

    PubMed

    Pacary, Emilie; Tixier, Emmanuelle; Coulet, Florence; Roussel, Simon; Petit, Edwige; Bernaudin, Myriam

    2007-07-01

    This study demonstrates that the Rho-kinase (ROCK) inhibitor, Y-27632, potentiates not only the effect of cobalt chloride (CoCl(2)) but also that of deferoxamine, another HIF-1 inducer, on mesenchymal stem cell (MSC) neuronal differentiation. HIF-1 is essential for CoCl(2)+/-Y-27632-induced MSC neuronal differentiation, since agents inhibiting HIF-1 abolish the changes of morphology and cell cycle arrest-related gene or protein expressions (p21, cyclin D1) and the increase of neuronal marker expressions (Tuj1, NSE). Y-27632 potentiates the CoCl(2)-induced decrease of cyclin D1 and nestin expressions, the increase of HIF-1 activation and EPO expression, and decreases pVHL expression. Interestingly, CoCl(2) decreases RhoA expression, an effect potentiated by Y-27632, revealing crosstalk between HIF-1 and RhoA/ROCK pathways. Moreover, we demonstrate a synergistic effect of CoCl(2) and Y-27632 on neurosphere differentiation into neurons and PC12 neurite outgrowth underlining that a co-treatment targeting both HIF-1 and ROCK pathways might be relevant to differentiate stem cells into neurons.

  10. Human placental eXpanded (PLX) mesenchymal-like adherent stromal cells confer neuroprotection to nerve growth factor (NGF)-differentiated PC12 cells exposed to ischemia by secretion of IL-6 and VEGF.

    PubMed

    Lahiani, Adi; Zahavi, Efrat; Netzer, Nir; Ofir, Racheli; Pinzur, Lena; Raveh, Shani; Arien-Zakay, Hadar; Yavin, Ephraim; Lazarovici, Philip

    2015-02-01

    Mesenchymal stem cells are potent candidates in stroke therapy due to their ability to secrete protective anti-inflammatory cytokines and growth factors. We investigated the neuroprotective effects of human placental mesenchymal-like adherent stromal cells (PLX) using an established ischemic model of nerve growth factor (NGF)-differentiated pheochromocytoma PC12 cells exposed to oxygen and glucose deprivation (OGD) followed by reperfusion. Under optimal conditions, 2 × 10⁵ PLX cells, added in a trans-well system, conferred 30-60% neuroprotection to PC12 cells subjected to ischemic insult. PC12 cell death, measured by LDH release, was reduced by PLX cells or by conditioned medium derived from PLX cells exposed to ischemia, suggesting the active release of factorial components. Since neuroprotection is a prominent function of the cytokine IL-6 and the angiogenic factor VEGF165, we measured their secretion using selective ELISA of the cells under ischemic or normoxic conditions. IL-6 and VEGF165 secretion by co-culture of PC12 and PLX cells was significantly higher under ischemic compared to normoxic conditions. Exogenous supplementation of 10 ng/ml each of IL-6 and VEGF165 to insulted PC12 cells conferred neuroprotection, reminiscent of the neuroprotective effect of PLX cells or their conditioned medium. Growth factors as well as co-culture conditioned medium effects were reduced by 70% and 20% upon pretreatment with 240 ng/ml Semaxanib (anti VEGF165) and/or 400 ng/ml neutralizing anti IL-6 antibody, respectively. Therefore, PLX-induced neuroprotection in ischemic PC12 cells may be partially explained by IL-6 and VEGF165 secretion. These findings may also account for the therapeutic effects seen in clinical trials after treatment with these cells.

  11. A novel neurotrophic property of glucagon-like peptide 1: a promoter of nerve growth factor-mediated differentiation in PC12 cells.

    PubMed

    Perry, TracyAnn; Lahiri, Debomoy K; Chen, Demao; Zhou, Jie; Shaw, Karen T Y; Egan, Josephine M; Greig, Nigel H

    2002-03-01

    The insulinotropic hormone glucagon-like peptide-1 (7-36)-amide (GLP-1) has potent effects on glucose-dependent insulin secretion, insulin gene expression, and pancreatic islet cell formation and is presently in clinical trials as a therapy for type 2 diabetes mellitus. We report on the effects of GLP-1 and two of its long-acting analogs, exendin-4 and exendin-4 WOT, on neuronal proliferation and differentiation, and on the metabolism of two neuronal proteins in the rat pheochromocytoma (PC12) cell line, which has been shown to express the GLP-1 receptor. We observed that GLP-1 and exendin-4 induced neurite outgrowth in a manner similar to nerve growth factor (NGF), which was reversed by coincubation with the selective GLP-1 receptor antagonist exendin (9-39). Furthermore, exendin-4 could promote NGF-initiated differentiation and may rescue degenerating cells after NGF-mediated withdrawal. These effects were induced in the absence of cellular dysfunction and toxicity as quantitatively measured by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays, respectively. Our findings suggest that such peptides may be used in reversing or halting the neurodegenerative process observed in neurodegenerative diseases, such as the peripheral neuropathy associated with type 2 diabetes mellitus and Alzheimer's and Parkinson's diseases. Due to its novel twin action, GLP-1 and exendin-4 have therapeutic potential for the treatment of diabetic peripheral neuropathy and these central nervous system disorders.

  12. Mitochondria Related Pathway Is Essential for Polysaccharides Purified from Sparassis crispa Mediated Neuro-Protection against Glutamate-Induced Toxicity in Differentiated PC12 Cells

    PubMed Central

    Hu, Shuang; Wang, Di; Zhang, Junrong; Du, Mengyan; Cheng, Yingkun; Liu, Yan; Zhang, Ning; Wang, Di; Wu, Yi

    2016-01-01

    The present study aims to explore the neuro-protective effects of purified Sparassis crispa polysaccharides against l-glutamic acid (l-Glu)-induced differentiated PC12 (DPC12) cell damages and its underlying mechanisms. The Sparassis crispa water extract was purified by a DEAE-52 cellulose anion exchange column and a Sepharose G-100 column. A fraction with a molecular weight of 75 kDa and a diameter of 88.9 nm, entitled SCWEA, was obtained. SCWEA was identified with a triple helix with (1→3)-linked Rha in the backbone, and (1→2) linkages and (1→6) linkages in the side bone. Our results indicated that the pre-treatment of DPC12 cells with SCWEA prior to l-Glu exposure effectively reversed the reduction on cell viability (by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay) and reduced l-Glu-induced apoptosis (by Hoechst staining). SCWEA decreased the accumulation of intracellular reactive oxygen species, blocked Ca2+ influx and prevented depolarization of the mitochondrial membrane potential in DPC12 cells. Furthermore, SCWEA normalized expression of anti-apoptotic proteins in l-Glu-explored DPC12 cells. These results suggested that SCWEA protects against l-Glu-induced neuronal apoptosis in DPC12 cells and may be a promising candidate for treatment against neurodegenerative disease. PMID:26821016

  13. Chronic exposure to sub-lethal beta-amyloid (Abeta) inhibits the import of nuclear-encoded proteins to mitochondria in differentiated PC12 cells.

    PubMed

    Sirk, Daniel; Zhu, Ziping; Wadia, Jehangir S; Shulyakova, Natalya; Phan, Nam; Fong, Jamie; Mills, Linda R

    2007-12-01

    Studies on amyloid beta (Abeta|), the peptide thought to play a crucial role in the pathogenesis of Alzheimer's disease, have implicated mitochondria in Abeta-mediated neurotoxicity. We used differentiated PC12 cells stably transfected with an inducible green fluorescent protein (GFP) fusion protein containing an N'-terminal mitochondrial targeting sequence (mtGFP), to examine the effects of sub-lethal Abeta on the import of nuclear-encoded proteins to mitochondria. Exposure to sub-lethal Abeta(25-35) (10 mumol/L) for 48 h inhibited mtGFP import to mitochondria; average rates decreased by 20 +/- 4%. Concomitant with the decline in mtGFP, cytoplasmic mtGFP increased significantly while mtGFP expression and intramitochondrial mtGFP turnover were unchanged. Sub-lethal Abeta(1-42) inhibited mtGFP import and increased cytoplasmic mtGFP but only after 96 h. The import of two endogenous nuclear-encoded mitochondrial proteins, mortalin/mtHsp70 and Tom20 also declined. Prior to the decline in import, mitochondrial membrane potential (mmp), and reactive oxygen species levels were unchanged in Abeta-treated cells versus reverse phase controls. Sustained periods of decreased import were associated with decreased mmp, increased reactive oxygen species, increased vulnerability to oxygen-glucose deprivation and altered mitochondrial morphology. These findings suggest that an Abeta-mediated inhibition of mitochondrial protein import, and the consequent mitochondrial impairment, may contribute to Alzheimer's disease.

  14. NAD+ treatment can prevent rotenone-induced increases in DNA damage, Bax levels and nuclear translocation of apoptosis-inducing factor in differentiated PC12 cells.

    PubMed

    Hong, Yunyi; Nie, Hui; Wei, Xunbin; Fu, Shen; Ying, Weihai

    2015-04-01

    Nicotinamide adenine dinucleotide (NAD(+)) plays critical roles in energy metabolism, mitochondrial functions, calcium homeostasis and immunological functions. Our previous studies have found that NAD(+) administration can profoundly decrease ischemic brain injury and traumatic brain injury. Our recent study has also provided first direct evidence indicating that NAD(+) treatment can decrease cellular apoptosis, while the mechanisms underlying this protective effect remain unclear. In our current study, we determined the effects of NAD(+) treatment on several major factors in apoptosis and necrosis, including levels of Bax and nuclear translocation of apoptosis-inducing factor (AIF), as well as levels of DNA double-strand breaks (DSBs) and intracellular ATP in rotenone-treated differentiated PC12 cells. We found that NAD(+) treatment can markedly attenuate the rotenone-induced increases in the levels of Bax and nuclear translocation of AIF in the cells. We further found that NAD(+) treatment can significantly attenuate the rotenone-induced increase in the levels of DSBs and decrease in the intracellular ATP levels. Collectively, our study has suggested mechanisms underlying the preventive effects of NAD(+) on apoptosis, which has highlighted the therapeutic potential of NAD(+) for decreasing apoptotic changes in multiple major diseases.

  15. Differential morphology and composition of inclusions in the R6/2 mouse and PC12 cell models of Huntington's disease.

    PubMed

    Wanderer, Jonathan; Morton, A Jennifer

    2007-05-01

    The histological hallmark feature of Huntington's disease (HD) and other polyglutamine repeat diseases is the presence of intracellular inclusions. Much work has been devoted to trying to determine the relationship between inclusion formation and neuronal injury. However, little attention has been paid to the variability and characteristics of inclusions themselves. Here, we characterize the morphological and biochemical composition of inclusions in both a transgenic mouse model (R6/2 line) and an inducible cell culture model of HD (iPC12Q74). We identified several morphologically distinct kinds of inclusions in different locations (nuclei, cytoplasm and cellular processes). Ubiquitin colocalized completely with all of these inclusions in both the iPC12Q72 and R6/2 models. In the inclusions in iPC12Q74 cells, the 20S and 11S proteasome subunits colocalized variably, and the 19S subunit did not colocalize at all. In inclusions in R6/2 mouse neurons, the 20S subunit colocalized completely, but neither the 11S nor the 19S subunits colocalized at all. While the role of inclusions in the pathogenesis of HD continues to be debated, we suggest that the content and structure of inclusions vary considerably, not only from cell to cell but even within individual cells. Their role in the pathogenesis of HD is likely to depend on their location as well as their composition.

  16. Aspartame-induced apoptosis in PC12 cells.

    PubMed

    Horio, Yukari; Sun, Yongkun; Liu, Chuang; Saito, Takeshi; Kurasaki, Masaaki

    2014-01-01

    Aspartame is an artificial sweetner added to many low-calorie foods. The safety of aspartame remains controversial even though there are many studies on its risks. In this study, to understand the physiological effects of trace amounts of artificial sweetners on cells, the effects of aspartame on apoptosis were investigated using a PC12 cell system. In addition, the mechanism of apoptosis induced by aspartame in PC12 cells and effects on apoptotic factors such as cytochrome c, apoptosis-inducing factor, and caspase family proteins were studied by Western blotting and RT-PCR. Aspartame-induced apoptosis in PC12 cells in a dose-dependent manner. In addition, aspartame exposure increased the expressions of caspases 8 and 9, and cytochrome c. These results indicate that aspartame induces apoptosis mainly via mitochondrial pathway involved in apoptosis due to oxigen toxicity.

  17. Isorhamnetin, A Flavonol Aglycone from Ginkgo biloba L., Induces Neuronal Differentiation of Cultured PC12 Cells: Potentiating the Effect of Nerve Growth Factor.

    PubMed

    Xu, Sherry L; Choi, Roy C Y; Zhu, Kevin Y; Leung, Ka-Wing; Guo, Ava J Y; Bi, Dan; Xu, Hong; Lau, David T W; Dong, Tina T X; Tsim, Karl W K

    2012-01-01

    Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, share a chemical resemblance to estrogen, and indeed some of which have been used as estrogen substitutes. In searching for possible functions of flavonoids, the neuroprotective effect in brain could lead to novel treatment, or prevention, for neurodegenerative diseases. Here, different subclasses of flavonoids were analyzed for its inductive role in neurite outgrowth of cultured PC12 cells. Amongst the tested flavonoids, a flavonol aglycone, isorhamnetin that was isolated mainly from the leaves of Ginkgo biloba L. showed robust induction in the expression of neurofilament, a protein marker for neurite outgrowth, of cultured PC12 cells. Although isorhamnetin by itself did not show significant inductive effect on neurite outgrowth of cultured PC12 cells, the application of isorhamnetin potentiated the nerve growth factor- (NGF-)induced neurite outgrowth. In parallel, the expression of neurofilaments was markedly increased in the cotreatment of NGF and isorhamnetin in the cultures. The identification of these neurite-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimer's disease and depression.

  18. Neuroprotective effects of dimerumic acid and deferricoprogen from Monascus purpureus NTU 568-fermented rice against 6-hydroxydopamine-induced oxidative stress and apoptosis in differentiated pheochromocytoma PC-12 cells.

    PubMed

    Tseng, Wei-Ting; Hsu, Ya-Wen; Pan, Tzu-Ming

    2016-08-01

    Context Oxidative stress plays a key role in neurodegenerative disorders, including Parkinson's disease (PD). Rice fermented with Monascus purpureus Went (Monascaceae) NTU 568 (red mould rice) was found to contain antioxidants, including dimerumic acid (DMA) and deferricoprogen (DFC). Objective The effects of DMA and DFC on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity and potential protective mechanisms in differentiated PC-12 pheochromocytoma cells were investigated. Materials and methods DMA (0-60 μM) or DFC (0-10 μM) was co-treated with 6-OHDA (200 μM, 24 h exposure) in differentiated PC-12 cells. Cell viability and intercellular reactive oxygen species (ROS) were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assays, respectively. Cell apoptosis was determined by DNA fragmentation analysis and propidium iodide staining by flow cytometry. Western blot analysis was used to measure the levels of cell protein expression. Results DMA and DFC significantly increased cell viability to 72% and 81% in 6-OHDA-induced differentiated PC-12 cell cultures, respectively. Furthermore, DMA and DFC reduced 6-OHDA-induced formation of extracellular and intercellular ROS by 25% and 20%, respectively, and decreased NADPH oxidase-2 expression in differentiated PC-12 cells. DMA and DFC inhibited 6-OHDA-induced apoptosis and decreased activation of caspase-3 via regulation of Bcl-2-associated X protein (Bax) and Bcl-2 protein expression in differentiated PC-12 cells. Conclusion DMA and DFC may protect against 6-OHDA toxicity by inhibiting ROS formation and apoptosis. These results showed that the metabolites from M. purpureus NTU 568 fermentation were potential therapeutic agents for PD induced by oxidative damage and should be encouraged for further research.

  19. Effect of morphine on PC12 cells with molecular radar

    NASA Astrophysics Data System (ADS)

    Shi, Chen; Yu, Xiaoli; Lu, Jiuyi; Zhang, Chunyang; Jin, Lei; Ma, Hui; Zhang, Dacheng; Chen, Die Yan

    2000-10-01

    Molecular Radar (MR) is a new method to detect biological processes in living cells at the level of molecular, it is also the newest means to get intracellular information. In this paper we study the effect of morphine on PC12 cells using MR. The results show that the effect of morphine on PC12 cells is time- and concentration-dependent. Morphine treating for short time induces the increase and fluctuation of intracellular (CA2+), while morphine treating for long time induces chromatin condensation, loss of mitochondria membrane potential apoptosis.

  20. Effects of eicosapentaenoic acid on synaptic plasticity, fatty acid profile and phosphoinositide 3-kinase signaling in rat hippocampus and differentiated PC12 cells.

    PubMed

    Kawashima, Akiko; Harada, Tsuyoshi; Kami, Hideaki; Yano, Takashi; Imada, Kazunori; Mizuguchi, Kiyoshi

    2010-04-01

    Placebo-controlled clinical studies suggest that intake of n-3 polyunsaturated fatty acids improves neurological disorders such as Alzheimer's disease, Huntington's disease and schizophrenia. To evaluate the impact of eicosapentaenoic acid (EPA), we orally administered highly purified ethyl EPA (EPA-E) to rats at a dose of 1.0 mg/g per day and measured long-term potentiation of the CA1 hippocampal region, a physiological correlate of synaptic plasticity that is thought to underlie learning and memory. The mean field excitatory postsynaptic potential slope of the EPA-E group was significantly greater than that of the control group in the CA1 region. Gene expression of hippocampal p85alpha, one of the regulatory subunits of phosphatidylinositol 3-kinase (PI3-kinase), was increased with EPA-E administration. Investigation of fatty acid profiles of neuronal and glia-enriched fractions demonstrated that a single administration of EPA-E significantly increased neuronal and glial EPA content and glial docosahexaenoic acid content, clearly suggesting that EPA was indeed taken up by both neurons and glial cells. In addition, we investigated the direct effects of EPA on the PI3-kinase/Akt pathway in differentiated PC12 cells. Phosphorylated-Akt expression was significantly increased in EPA-treated cells, and nerve growth factor withdrawal-induced increases in cell death and caspase-3 activity were suppressed by EPA treatment. These findings suggest that EPA protects against neurodegeneration by modulating synaptic plasticity and activating the PI3-kinase/Akt pathway, possibly by its own functional effects in neurons and glial cells and by its capacity to increase brain docosahexaenoic acid.

  1. Induction of cytoprotective autophagy in PC-12 cells by cadmium

    SciTech Connect

    Wang, Qiwen; Zhu, Jiaqiao; Zhang, Kangbao; Jiang, Chenyang; Wang, Yi; Yuan, Yan; Bian, Jianchun; Liu, Xuezhong; Gu, Jianhong; Liu, Zongping

    2013-08-16

    Highlights: •Cadmium can promote early upregulation of autophagy in PC-12 cells. •Autophagy precedes apoptosis in cadmium-treated PC-12 cells. •Cadmium-induced autophagy is cytoprotective in PC-12 cells. •Class III PI3K/beclin-1/Bcl-2 signaling pathway plays a positive role in cadmium-triggered autophagy. -- Abstract: Laboratory data have demonstrated that cadmium (Cd) may induce neuronal apoptosis. However, little is known about the role of autophagy in neurons. In this study, cell viability decreased in a dose- and time-dependent manner after treatment with Cd in PC-12 cells. As cells were exposed to Cd, the levels of LC3-II proteins became elevated, specific punctate distribution of endogenous LC3-II increased, and numerous autophagosomes appeared, which suggest that Cd induced a high level of autophagy. In the late stages of autophagy, an increase in the apoptosis ratio was observed. Likewise, pre-treatment with chloroquine (an autophagic inhibitor) and rapamycin (an autophagic inducer) resulted in an increased and decreased percentage of apoptosis in contrast to other Cd-treated groups, respectively. The results indicate that autophagy delayed apoptosis in Cd-treated PC-12 cells. Furthermore, co-treatment of cells with chloroquine reduced autophagy and cell activity. However, rapamycin had an opposite effect on autophagy and cell activity. Moreover, class III PI3 K/beclin-1/Bcl-2 signaling pathways served a function in Cd-induced autophagy. The findings suggest that Cd can induce cytoprotective autophagy by activating class III PI3 K/beclin-1/Bcl-2 signaling pathways. In sum, this study strongly suggests that autophagy may serve a positive function in the reduction of Cd-induced cytotoxicity.

  2. Dystrophin Dp71 in PC12 cell adhesion

    PubMed Central

    Enríquez-Aragón, Jose Arturo; Cerna-Cortés, Joel; Bermúdez de León, Mario; García-Sierra, Francisco; González, Everardo; Mornet, Dominique; Cisneros, Bulmaro

    2005-01-01

    Previously, we reported that PC12 cells with decreased Dp71 expression (antisense-Dp71 cells) display deficient nerve-growth-factor-induced neurite outgrowth. In this study, we show that disturbed neurite outgrowth of antisense-Dp71 cells is accompanied by decreased adhesion activity on laminin, collagen and fibronectin. In wild-type cells, the immunostaining of Dp71 and _1-integrin overlaps in the basal area contacting the substrate, but staining of both proteins decrease in the antisense-Dp71 cells. Morphology of antisense-Dp71 cells at the electron microscopic level is characterized by the lack of filopodia, cellular projections involved in adhesion. Our findings suggest that Dp71 is required for the efficient PC12 cell attachment to b1-integrin-dependent substrata and that decreased adhesion activity of the anti-sense-Dp71 cells could determine their deficiency to extend neurites. PMID:15706226

  3. Cell cycle markers have different expression and localization patterns in neuron-like PC12 cells and primary hippocampal neurons.

    PubMed

    Negis, Yesim; Unal, Aysegul Yildiz; Korulu, Sirin; Karabay, Arzu

    2011-06-01

    Neuron-like PC12 cells are extensively used in place of neurons in published studies. Aim of this paper has been to compare mRNA and protein expressions of cell cycle markers; cyclinA, B, D, E; Cdk1, 2 and 4; and p27 in post-mitotic primary hippocampal neurons, mitotically active PC12 cells and NGF-differentiated post-mitotic PC12 cells. Contrary to PC12 cells, in neurons, the presence of all these markers was detected only at mRNA level; except for cyclinA, cyclinE and Cdk4, which were detectable also at protein levels. In both NGF-treated PC12 cells and neurons, cyclinE was localized only in the nucleus. In NGF-treated PC12 cells cyclinD and Cdk4 were localized in the nucleus while, in neurons cyclinD expression was not detectable; Cdk4 was localized in the cytoplasm. In neurons, cyclinA was nuclear, whereas in NGF-treated PC12 cells, it was localized in the cell body and along the processes. These results suggest that PC12 cells and primary neurons are different in terms of cell cycle protein expressions and localizations. Thus, it may not be very appropriate to use these cells as neuronal model system in order to understand neuronal physiological activities, upstream of where may lie cell cycle activation triggered events.

  4. Gab1 mediates neurite outgrowth, DNA synthesis, and survival in PC12 cells.

    PubMed

    Korhonen, J M; Saïd, F A; Wong, A J; Kaplan, D R

    1999-12-24

    The Gab1-docking protein has been shown to regulate phosphatidylinositol 3-kinase PI3K activity and potentiate nerve growth factor (NGF)-induced survival in PC12 cells. Here, we investigated the potential of Gab1 to induce neurite outgrowth and DNA synthesis, two other important aspects of NGF-induced neuronal differentiation of PC12 cells and NGF-independent survival. We generated a recombinant adenovirus encoding hemagglutinin (HA)-epitope-tagged Gab1 and expressed this protein in PC12 cells. HA-Gab1 was constitutively tyrosine-phosphorylated in PC12 cells and induced the phosphorylation of Akt/protein kinase B and p44/42 mitogen-activated protein kinase. HA-Gab1-stimulated a 10-fold increase in neurite outgrowth in the absence of NGF and a 5-fold increase in NGF-induced neurite outgrowth. HA-Gab1 also stimulated DNA synthesis and caused NGF-independent survival in PC12 cells. Finally, we found that HA-Gab1-induced neuritogenesis was completely suppressed by pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activity and 50% suppressed by inhibition of PI3K activity. In contrast, HA-Gab1-stimulated cell survival was efficiently suppressed only by inhibition of both PI3K and MEK activities. These results indicate that Gab1 is capable of mediating differentiation, DNA synthesis, and cell survival and uses both PI3K and MEK signaling pathways to achieve its effects.

  5. Functionality of NGF-protected PC12 cells following exposure to 6-hydroxydopamine

    SciTech Connect

    Kavanagh, Edel T.; Loughlin, John P.; Herbert, Kate Reed; Dockery, Peter; Samali, Afshin; Doyle, Karen M.; Gorman, Adrienne M. . E-mail: adrienne.gorman@nuigalway.ie

    2006-12-29

    6-Hydroxydopamine (6-OHDA) is often used in models of Parkinson's disease since it can selectively target and kill dopaminergic cells of the substantia nigra. In this study, pre-treatment of PC12 cells with nerve growth factor (NGF) inhibited apoptosis and necrosis by 6-OHDA, including caspase activity and lactate dehydrogenase release. Notably, cells exposed to 6-OHDA in the presence of NGF were subsequently capable of proliferation (when replated without NGF), or neurite outgrowth (with continued presence of NGF). Following 7 days growth in the presence of NGF, expression of {beta}III tubulin and tyrosine hydroxylase and increased intracellular catecholamines was detectable in PC12 cells, features characteristic of functional dopaminergic neurons. NGF-pre-treated PC12 cells retained expression of {beta}III-tubulin and tyrosine hydroxylase, but not catecholamine content following 6-OHDA exposure. These data indicate that NGF-protected cells maintained some aspects of functionality and were subsequently capable of proliferation or differentiation.

  6. Establishment and characterization of methylmercury-resistant PC12 cell line.

    PubMed Central

    Miura, K; Clarkson, T W; Ikeda, K; Naganuma, A; Imura, N

    1994-01-01

    Methylmercury (MeHg)-resistant sublines of rat pheochromocytoma (PC12) cells were isolated by repeated exposure to stepwise increased concentrations of MeHg. One of the sublines (PC12/TM) showed an 8- to 10-fold increase in resistance to MeHg compared with parent PC12 cells on the basis of the concentration required for 50% inhibition (IC50) of growth. PC12/TM cells accumulated smaller amounts of MeHg than parent PC12 cells. This reduction in MeHg accumulation in PC12/TM cells resulted from slow uptake and rapid efflux. The intracellular glutathione (GSH) level in PC12/TM cells was four times higher than that of PC12 cells. Pretreatment of PC12/TM cells with buthionine sulfoximine, which decreased the GSH level to that of the parent PC12 cells, increased the sensitivity of PC12/TM cells to MeHg. A close correlation between the MeHg accumulation and MeHg sensitivity was found among seven sublines of PC12 cells and parent PC12 cell line. The GSH level in PC12 sublines was also correlated with their sensitivity to MeHg. PMID:7843125

  7. A Novel Ligustrazine Derivative T-VA Prevents Neurotoxicity in Differentiated PC12 Cells and Protects the Brain against Ischemia Injury in MCAO Rats

    PubMed Central

    Li, Guoling; Tian, Yufei; Zhang, Yuzhong; Hong, Ying; Hao, Yingzhi; Chen, Chunxiao; Wang, Penglong; Lei, Haimin

    2015-01-01

    Broad-spectrum drugs appear to be more promising for the treatment of acute ischemic stroke. In our previous work, a new ligustrazine derivative (3,5,6-trimethylpyrazin-2-yl) methyl 3-methoxy-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate (T-VA) showed neuroprotective effect on injured PC12 cells (EC50 = 4.249 µM). In the current study, we show that this beneficial effect was due to the modulation of nuclear transcription factor-κB/p65 (NF-κB/p65) and cyclooxygenase-2 (COX-2) expressions. We also show that T-VA exhibited neuroprotective effect in a rat model of ischemic stroke with concomitant improvement of motor functions. We propose that the protective effect observed in vivo is owing to increased vascular endothelial growth factor (VEGF) expression, decreased oxidative stress, and up-regulation of Ca2+–Mg2+ ATP enzyme activity. Altogether, our results warrant further studies on the utility of T-VA for the potential treatment of ischemic brain injuries, such as stroke. PMID:26370988

  8. Effects of Extremely Low Frequency Magnetic Field on Neurite Outgrowth of PC12 and PC12D Cells and Evaluation by Image Analysis

    NASA Astrophysics Data System (ADS)

    Sakanishi, Akio; Takatsuki, Hideyo; Yoshikoshi, Akio; Fujiwara, Yasuyoshi

    2004-05-01

    A pheochromocytoma cell (PC12), and its derivative (PC12D), differentiate to nervelike cells in culture with the nerve growth factor (NGF) and forskolin respectively. We introduced a morphological factor σ=L/2(π A)1/2 for quantitating neurite outgrowth under a microscope in the presence of extremely low-frequency (ELF) magnetic fields for 22 hours, where L and A are the contour length and the area of the cells in clump determined using an image-analysis system. ELF magnetic fields B1 were generated with a single coil or double coils in Helmholtz configuration together with static fields B0 of -53, -20 and 67 μT. σ increased with increasing NGF or forskolin level at B0=-53 μT (geomagnetism), in agreement with the cytometric observation of micrographs. With the addition of an AC field B1 at 60 Hz (100 μT > B1 > 3 μT rms) to B0, neurite outgrowth represented by σ was depressed for PC12 and stimulated for PC12D. We discuss the cyclotron resonance and the ion parametric resonance models.

  9. Habituation in the Single Cell: Diminished Secretion of Norepinephrine with Repetitive Depolarization of PC12 Cells

    NASA Astrophysics Data System (ADS)

    McFadden, Philip N.; Koshland, Daniel E., Jr.

    1990-03-01

    Neuronally differentiated PC12 cells secrete decreasing amounts of [^3H]norepinephrine when repetitively stimulated by depolarizing concentrations of potassium ion. The decreasing response shows attributes that have been classically ascribed to response habituation, a behavior commonly observed in nervous systems but found here in a homogeneous cell type. Alteration of the habituation pattern was caused by activators of the protein kinase C pathway and of voltage-gated calcium channels.

  10. DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells.

    PubMed

    Won, Jong Hoon; Ahn, Kyong Hoon; Back, Moon Jung; Ha, Hae Chan; Jang, Ji Min; Kim, Ha Hyung; Choi, Sang-Zin; Son, Miwon; Kim, Dae Kyong

    2015-01-01

    In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.

  11. Microconical silicon structures influence NGF-induced PC12 cell morphology.

    PubMed

    Simitzi, C; Stratakis, E; Fotakis, C; Athanassakis, I; Ranella, A

    2015-04-01

    Micro-and nanofabrication techniques provide the opportunity to develop new types of cell culture platform, where the effect of various topographical cues on cellular functions such as proliferation and differentiation can be studied. In this study, PC12 cells were cultured on patterned silicon (Si) surfaces comprising arrays of microcones (MCs) exhibiting different geometrical characteristics and surface chemistries. It was illustrated that, in the absence of nerve growth factor (NGF), PC12 cells increased proliferation on all types of patterned surface, as compared to flat Si surfaces. However, in the presence of NGF, PC12 cells showed different responses, depending on the plating surface. Unlike low and intermediate rough MC surfaces, highly rough ones exhibiting large distances between MCs did not support PC12 cell differentiation, independently of the MCs' chemical coatings. These results suggest that the geometrical characteristics of MCs alone can influence specific cellular functions. Tailoring of the physical properties of arrays of Si MCs in order to identify which combinations of MC topologies and spatially defined chemistries are capable of driving specific cellular responses is envisaged.

  12. Curcumin Protects β-Lactoglobulin Fibril Formation and Fibril-Induced Neurotoxicity in PC12Cells

    PubMed Central

    Mazaheri, Mansooreh; Moosavi-Movahedi, Ali Akbar; Saboury, Ali Akbar; Khodagholi, Fariba; Shaerzadeh, Fatemeh; Sheibani, Nader

    2015-01-01

    In this study the β-lactoglobulin fibrillation, in the presence or absence of lead ions, aflatoxin M1 and curcumin, was evaluated using ThT fluorescence, Circular dichroism spectroscopy and atomic force microscopy. To investigate the toxicity of the different form of β-Lg fibrils, in the presence or absence of above toxins and curcumin, we monitored changes in the level of reactive oxygen species and morphology of the differentiated neuron-like PC12 cells. The cell viability, cell body area, average neurite length, neurite width, number of primary neurites, percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different form of β-Lg fibrils. Incubation of β-Lg with curcumin resulted in a significant decrease in ROS levels even in the presence of lead ions and aflatoxin M1. The β-Lg fibrils formed in the presence of lead ions and aflatoxin M1 attenuated the growth and complexity of PC12 cells compared with other form of β-Lg fibrils. However, the adverse effects of these toxins and protein fibrils were negated in the presence of curcumin. Furthermore, the antioxidant and inhibitory effects of curcumin protected PC12 cells against fibril neurotoxicity and enhanced their survival. Thus, curcumin may provide a protective effect toward β-Lg, and perhaps other protein, fibrils mediated neurotoxicity. PMID:26186474

  13. Neurosecretory Habituation in PC12 Cells: Modulation During Parallel Habituation

    NASA Astrophysics Data System (ADS)

    Martin, Paul T.; Koshland, Daniel E., Jr.

    1995-05-01

    PC12 cells habituate during repetitive stimulation with acetylcholine, bradykinin, or high potassium. Interspersing these stimulants did not affect the rate of habituation of the others, but it could modulate the amplitude of the norepinephrine secretion each could achieve. Stimulation with acetylcholine inhibited norepinephrine secretion caused by high potassium and bradykinin stimulation, while high potassium had no effect on acetylcholine or bradykinin, and bradykinin increased secretion caused by acetylcholine. Changes in norepinephrine secretion resulting from any of these stimulants correlated with changes in internal calcium levels. Cyclic AMP-, protein kinase C-, and calmodulin-dependent second messenger pathways all modulated norepinephrine secretion caused by acetylcholine and high potassium and showed a distinct hierarchy in their effectiveness. These data demonstrate that different receptor pathways can change the norepinephrine response of one another while not changing the levels of the molecules responsible for habituation.

  14. Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2

    PubMed Central

    2013-01-01

    Background Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense’ and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified. Results To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition. Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2. Conclusion Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties. PMID:24119372

  15. Quercetin-3-O-(2″-galloyl)-α-L-rhamnopyranoside attenuates cholesterol oxidation product-induced apoptosis by suppressing NF-κB-mediated cell death process in differentiated PC12 cells.

    PubMed

    Lee, Da Hee; Nam, Yoon Jeong; Lee, Chung Soo

    2015-08-01

    Cholesterol oxidation products are suggested to be involved in neuronal cell degeneration. We examined the preventive effect of quercetin-3-O-(2″-galloyl)-α-L-rhamnopyranoside (QGR), a quercetin derivative, on the cholesterol oxidation product-induced neuronal cell death using differentiated PC12 cells in relation to nuclear factor (NF)-κB-mediated apoptotic process. 7-Ketocholesterol and 25-hydroxycholesterol induced a decrease in the levels of BH3 interacting-domain death agonist (Bid) and B cell lymphoma 2 (Bcl-2), increase in the levels of Bcl-2-associated X protein (Bax) and p53, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases, and cleavage of poly(ADP-ribose) polymerase 1 (PARP-1). 7-Ketocholesterol induced increase in cytosolic and nuclear NF-κB p65, nuclear phospho-NF-κB p65, cytosolic NF-κB p50, and cytosolic phospho-IκB-α levels. The addition of QGR, N-acetyl cysteine, or Bay 11-7085 attenuated the cholesterol oxidation product-induced changes in the apoptosis-related protein levels, activation of NF-κB, formation of reactive oxygen species, depletion of glutathione (GSH), nuclear damage, and cell death. The results show that QGR may attenuate the cholesterol oxidation product-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways that is mediated by NF-κB activation. The preventive effect appears to be associated with the inhibitory effect on the formation of reactive oxygen species and depletion of GSH.

  16. Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells.

    PubMed Central

    Gutacker, C; Klock, G; Diel, P; Koch-Brandt, C

    1999-01-01

    Clusterin (apolipoprotein J) is an extracellular glycoprotein that might exert functions in development, cell death and lipid transport. Clusterin gene expression is elevated at sites of tissue remodelling, such as differentiation and apoptosis; however, the signals responsible for this regulation have not been identified. We use here the clusterin gene as a model system to examine expression in PC12 cells under the control of differentiation and proliferation signals produced by nerve growth factor (NGF) and by epidermal growth factor (EGF) respectively. NGF induced clusterin mRNA, which preceded neurite outgrowth typical of neuronal differentiation. EGF also activated the clusterin mRNA, demonstrating that both proliferation and differentiation signals regulate the gene. To localize NGF- and EGF-responsive elements we isolated the clusterin promoter and tested it in PC12 cell transfections. A 2.5 kb promoter fragment and two 1.5 and 0.3 kb deletion mutants were inducible by NGF and EGF. The contribution to this response of a conserved activator protein 1 (AP-1) motif located in the 0.3 kb fragment was analysed by mutagenesis. The mutant promoter was not inducible by NGF or EGF, which identifies the AP-1 motif as an element responding to both factors. Binding studies with PC12 nuclear extracts showed that AP-1 binds to this sequence in the clusterin promoter. These findings suggest that NGF and EGF, which give differential gene regulation in PC12 cells, resulting in neuronal differentiation and proliferation respectively, use the common Ras/extracellular signal-regulated kinase/AP-1 signalling pathway to activate clusterin expression. PMID:10215617

  17. KCl stimulation increases norepinephrine transporter function in PC12 cells.

    PubMed

    Mandela, Prashant; Ordway, Gregory A

    2006-09-01

    The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.

  18. Methylmercury decreases NGF-induced TrkA autophosphorylation and neurite outgrowth in PC12 cells.

    PubMed

    Parran, Damani K; Barone, Stanley; Mundy, William R

    2003-03-14

    Neurotrophin signaling through Trk receptors is important for differentiation and survival in the developing nervous system. The present study examined the effects of CH(3)Hg on (125)I-nerve growth factor (NGF) binding to the TrkA receptor, NGF-induced activation of the TrkA receptor, and neurite outgrowth in an in vitro model of differentiation using PC12 cells. Whole-cell binding assays using (125)I-NGF revealed a single binding site with a K(d) of approximately 1 nM. Methylmercury (CH(3)Hg) at 30 nM (EC(50) for neurite outgrowth inhibition) did not affect NGF binding to TrkA. TrkA autophosphorylation was measured by immunoblotting with a phospho-specific antibody. TrkA autophosphorylation peaked between 2.5 and 5 min of exposure and then decreased but was still detectable at 60 min. Concurrent exposure to CH(3)Hg and NGF for 2.5 min resulted in a concentration-dependent decrease in TrkA autophosphorylation, which was significant at 100 nM CH(3)Hg. To determine whether the observed inhibition of TrkA was sufficient to alter cell differentiation, NGF-stimulated neurite outgrowth was examined in PC12 cells after exposure to 30 nM CH(3)Hg, a concentration that inhibited TrkA autophosphorylation by approximately 50%. For comparison, a separate group of PC12 cells were exposed to a concentration of the selective Trk inhibitor K252a (30 nM), which had been shown to produce significant inhibition of TrkA autophosphorylation. Twenty-four hour exposure to either CH(3)Hg or K252a reduced neurite outgrowth to a similar degree. Our results suggest that CH(3)Hg may inhibit differentiation of PC12 cells by interfering with NGF-stimulated TrkA autophosphorylation.

  19. Platycodin D induced apoptosis and autophagy in PC-12 cells through mitochondrial dysfunction pathway

    NASA Astrophysics Data System (ADS)

    Zeng, Chuan-Chuan; Zhang, Cheng; Yao, Jun-Hua; Lai, Shang-Hai; Han, Bing-Jie; Li, Wei; Tang, Bing; Wan, Dan; Liu, Yun-Jun

    2016-11-01

    In this article, the in vitro cytotoxicity of platycodin D was evaluated in human PC-12, SGC-7901, BEL-7402, HeLa and A549 cancer cell lines. PC-12 cells were sensitive to platycodin D treatment, with an IC50 value of 13.5 ± 1.2 μM. Morphological and comet assays showed that platycodin D effectively induced apoptosis in PC-12 cells. Platycodin D increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Platycodin D induced cell cycle arrest at the G0/G1 phase in the PC-12 cell line. Platycodin D can induce autophagy. In addition, platycodin D can down-regulate the expression of Bcl-2 and Bcl-x, and up-regulate the levels of Bid protein in the PC-12 cells. The results demonstrated that platycodin D induced PC-12 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

  20. Induction of Neurite Outgrowth in PC12 Cells Treated with Temperature-Controlled Repeated Thermal Stimulation

    PubMed Central

    Kudo, Tada-aki; Kanetaka, Hiroyasu; Mochizuki, Kentaro; Tominami, Kanako; Nunome, Shoko; Abe, Genji; Kosukegawa, Hiroyuki; Abe, Toshihiko; Mori, Hitoshi; Mori, Kazumi; Takagi, Toshiyuki; Izumi, Shin-ichi

    2015-01-01

    To promote the functional restoration of the nervous system following injury, it is necessary to provide optimal extracellular signals that can induce neuronal regenerative activities, particularly neurite formation. This study aimed to examine the regulation of neuritogenesis by temperature-controlled repeated thermal stimulation (TRTS) in rat PC12 pheochromocytoma cells, which can be induced by neurotrophic factors to differentiate into neuron-like cells with elongated neurites. A heating plate was used to apply thermal stimulation, and the correlation of culture medium temperature with varying surface temperature of the heating plate was monitored. Plated PC12 cells were exposed to TRTS at two different temperatures via heating plate (preset surface temperature of the heating plate, 39.5°C or 42°C) in growth or differentiating medium for up to 18 h per day. We then measured the extent of growth, neuritogenesis, or acetylcholine esterase (AChE) activity (a neuronal marker). To analyze the mechanisms underlying the effects of TRTS on these cells, we examined changes in intracellular signaling using the following: tropomyosin-related kinase A inhibitor GW441756; p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 with its inactive analog, U0124, as a control. While a TRTS of 39.5°C did not decrease the growth rate of cells in the cell growth assay, it did increase the number of neurite-bearing PC12 cells and AChE activity without the addition of other neuritogenesis inducers. Furthermore, U0126, and SB203580, but not U0124 and GW441756, considerably inhibited TRTS-induced neuritogenesis. These results suggest that TRTS can induce neuritogenesis and that participation of both the ERK1/2 and p38 MAPK signaling pathways is required for TRTS-dependent neuritogenesis in PC12 cells. Thus, TRTS may be an effective technique for regenerative neuromedicine. PMID:25879210

  1. Synergistic effect of topography, surface chemistry and conductivity of the electrospun nanofibrous scaffold on cellular response of PC12 cells.

    PubMed

    Tian, Lingling; Prabhakaran, Molamma P; Hu, Jue; Chen, Menglin; Besenbacher, Flemming; Ramakrishna, Seeram

    2016-09-01

    Electrospun nanofibrous nerve implants is a promising therapy for peripheral nerve injury, and its performance can be tailored by chemical cues, topographical features as well as electrical properties. In this paper, a surface modified, electrically conductive, aligned nanofibrous scaffold composed of poly (lactic acid) (PLA) and polypyrrole (Ppy), referred to as o-PLAPpy_A, was fabricated for nerve regeneration. The morphology, surface chemistry and hydrophilicity of nanofibers were characterized by Scanning Electron Microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and water contact angle, respectively. The effects of these nanofibers on neuronal differentiation using PC12 cells were evaluated. A hydrophilic surface was created by Poly-ornithine coating, which was able to provide a better environment for cell attachment, and furthermore aligned fibers were proved to be able to guide PC12 cells grow along the fiber direction and be beneficial for neurite outgrowth. The cellular response of PC12 cells to pulsed electrical stimulation was evaluated by NF 200 and alpha tubulin expression, indicating that electrical stimulation with a voltage of 40mV could enhance the neurite outgrowth. The PC12 cells stimulated with electrical shock showed greater level of neurite outgrowth and smaller cell body size. Moreover, the PC12 cells under electrical stimulation showed better viability. In summary, the o-PLAPpy_A nanofibrous scaffold supported the attachment, proliferation and differentiation of PC12 cells in the absence of electrical stimulation, which could be potential candidate for nerve regeneration applications.

  2. Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

    PubMed Central

    Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R.; Millard, Ronald W.; Olah, Mark E.; Sankovic, John M.; Banerjee, Rupak K.

    2008-01-01

    Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions. PMID:19081771

  3. Panaxynol induces neurite outgrowth in PC12D cells via cAMP- and MAP kinase-dependent mechanisms.

    PubMed

    Wang, Ze-Jian; Nie, Bao-Ming; Chen, Hong-Zhuan; Lu, Yang

    2006-01-05

    Panaxynol, a polyacetylene ((3R)-heptadeca-1,9-diene-4,6-diyn-3-ol; syn. falcarinol), was isolated from the lipophilic fractions of Panax notoginseng, a Chinese traditional medicinal plant. In the present study, we reported the neurotrophic effects of panaxynol on PC12D cells and mechanism involved in neurite outgrowth of the cells. Panaxynol could morphologically promote neurite outgrowth in PC12D cells, concentration-dependently reduce cell division and up-regulate molecular marker (MAP1B) expression in PC12D cells. Panaxynol induces the elevation of intracellular cAMP in PC12D cells. The neurite outgrowth in PC12D cells induced by panaxynol could be inhibited by the protein kinase A inhibitor RpcAMPS and by MAP kinase kinase 1/2 inhibitor U0126. These observations reveal that panaxynol could induce the differentiation of PC12D cells in a process similar to but distinct from that of NGF and the panaxynol's effects were via cAMP- and MAP kinase-dependent mechanisms.

  4. Muscarinic activation of mitogen-activated protein kinase in PC12 cells.

    PubMed

    Berkeley, J L; Levey, A I

    2000-08-01

    Muscarinic acetylcholine receptors (mAChRs) activate many downstream signaling pathways, some of which can lead to mitogen-activated protein kinase (MAPK) phosphorylation and activation. MAPKs play roles in regulating cell growth, differentiation, and synaptic plasticity. Here, the activation of MAPK was examined in PC12 cells endogenously expressing mAChRs. Western blot analysis using a phosphospecific MAPK antibody revealed a dose-dependent and atropine-sensitive increase in MAPK phosphorylation in cells stimulated with carbachol (CCh). The maximal response occurred after 5 min and was rapidly reduced to baseline. To investigate the receptors responsible for CCh activation of MAPK in PC12 cells, the mAChR subtypes present were determined using RT-PCR and immunoprecipitation. RT-PCR was used to amplify fragments of the appropriate sizes for m1, m4, and m5, and the identities of the bands were confirmed with restriction digests. Immunoprecipitation using subtype-specific antibodies showed that approximately 95% of the expressed receptors were m4, whereas the remaining approximately 5% were m1 and m5. A highly specific m1 toxin completely blocked MAPK phosphorylation in response to CCh stimulation. The mAChR-induced MAPK activation was abolished by protein kinase C down-regulation and partially inhibited by pertussis toxin. Although m1 represents a small proportion of the total mAChR population, pharmacological evidence suggests that m1 is responsible for MAPK activation in PC12 cells.

  5. Induction of neuritogenesis in PC12 cells by a pulsed electromagnetic field via MEK-ERK1/2 signaling.

    PubMed

    Kudo, Tada-aki; Kanetaka, Hiroyasu; Shimizu, Yoshinaka; Abe, Toshihiko; Mori, Hitoshi; Mori, Kazumi; Suzuki, Eizaburo; Takagi, Toshiyuki; Izumi, Shin-ichi

    2013-01-01

    We examined the regulation of neuritogenesis by a pulsed electromagnetic field (PEMF) in rat PC12 pheochromocytoma cells, which can be induced to differentiate into neuron-like cells with elongated neurites by inducers such as nerve growth factor (NGF). Plated PC12 cells were exposed to a single PEMF (central magnetic flux density, 700 mT; frequency, 0.172 Hz) for up to 12 h per day and were then evaluated for extent of neuritogenesis or acetylcholine esterase (AChE) activity. To analyze the mechanism underlying the effect of the PEMF on the cells, its effects on intracellular signaling were examined using the ERK kinase (MEK) inhibitors PD098059 and U0126 (U0124 was used as a negative control for U0126). The number of neurite-bearing PC12 cells and AChE activity increased after PEMF exposure without the addition of other inducers of neuritogenesis. Additionally, PEMF exposure induced sustained activation of ERK1/2 in PC12 cells, but not in NR8383 rat alveolar macrophages. Furthermore, U0126 strongly inhibited PEMF-dependent ERK1/2 activation and neuritogenesis. The PEMF-dependent neuritogenesis was also suppressed by PD098059, but not U0124. These results suggest that PEMF stimulation independently induced neuritogenesis and that activation of MEK-ERK1/2 signaling was induced by a cell-type-dependent mechanism required for PEMF-dependent neuritogenesis in PC12 cells.

  6. Development of an ischemic tolerance model in a PC12 cell line

    PubMed Central

    Maric, Dragan; Ruetzler, Christl; Barker, Jeffery L; Hallenbeck, John M

    2006-01-01

    Although ischemic tolerance has been described in a variety of primary cell culture systems, no similar in vitro models have been reported with any cell line. A model of ischemic preconditioning in the rat pheochromocytoma PC12 cell line is described here. When compared to nonpreconditioned cells, preexposure of PC12 cells to 6 hours of oxygen and glucose deprivation (OGD) significantly increased cell viability after 15 hours of OGD 24 hours later. Flow cytometry analysis of cells labeled with specific markers for apoptosis, Annexin V, and Hoechst 33342, and of DNA content, revealed that apoptosis is involved in OGD-induced PC12 cell death and that preconditioning of the cells mainly counteracts the effect of apoptosis. Immunocytochemistry of caspase-3, a central executioner in the apoptotic process, further confirmed the activation of apoptotic pathways in OGD-induced PC12 cell death. This model may be useful to investigate the cellular mechanisms involved in neuronal transient tolerance following ischemia. PMID:15647748

  7. Enhanced neurite outgrowth of PC-12 cells on graphene-monolayer-coated substrates as biomimetic cues

    NASA Astrophysics Data System (ADS)

    Lee, Jong Ho; Shin, Yong Cheol; Jin, Oh Seong; Han, Dong-Wook; Kang, Seok Hee; Hong, Suck Won; Kim, Jong Man

    2012-11-01

    Neurons are electrically excitable cells that transmit and process information in the nervous system. Recently, the differentiation of human neural stem cells to neurons has been shown to be enhanced on graphene substrates, and differentiated neurons have been shown to be able to still carry electrical signals when stimulated by graphene electrodes. Graphene films grown by using chemical vapor deposition were transferred onto glass coverslips by using the scooping method and were then coated with fetal bovine serum for a neuronal cell culture. The graphene substrates as biomimetic cues have been shown to enhance the neurite outgrowth of PC-12 cells. Our findings suggest that graphene has a unique surface property that can promote neuronal cells, which should open tremendous opportunities in neuroscience, neural engineering and regenerative medicine.

  8. Baicalin inhibits colistin sulfate-induced apoptosis of PC12 cells.

    PubMed

    Jiang, Hong; Lv, Pengfei; Li, Jichang; Wang, Hongjun; Zhou, Tiezhong; Liu, Yingzi; Lin, Wei

    2013-10-05

    Baicalin, a type of flavonoid extracted from the dried root of Scutellaria baicalensis georgi, has been shown to effectively inhibit cell apoptosis. Therefore, we assumed that baicalin would suppress co-listin sulfate-induced neuronal apoptosis. PC12 cells exposed to colistin sulfate (62.5-500 μg/mL) for 24 hours resulted in PC12 cell apoptosis. In addition, caspase-3 activity, lactate dehydrogenase level and free radical content increased in a dose-dependent manner. Subsequently, PC12 cells were pretreated with baicalin (25, 50 and 100 μg/mL), and exposed to 125 μg/mL colistin sulfate. Cell morphology markedly changed, and cell viability increased. Moreover, caspase-3 activity, tate dehydrogenase level and free radical content decreased. Results indicated that baicalin inhi-bited colistin sulfate-induced PC12 cell apoptosis by suppressing free radical injury, and reducing caspase-3 activity and lactate dehydrogenase activity.

  9. Antibody-mediated inhibition of Nogo-A signaling promotes neurite growth in PC-12 cells

    PubMed Central

    Yazdi, Iman K; Taghipour, Nima; Hmaidan, Sarah; Palomba, Roberto; Scaria, Shilpa; Munoz, Alvaro; Boone, Timothy B; Tasciotti, Ennio

    2016-01-01

    The use of a monoclonal antibody to block the neurite outgrowth inhibitor Nogo-A has been of great interest for promoting axonal recovery as a treatment for spinal cord injury. While several cellular and non-cellular assays have been developed to quantify the bioactive effects of Nogo-A signaling, demand still exists for the development of a reliable approach to characterize the effectiveness of the anti-Nogo-A antibody. In this study, we developed and validated a novel cell-based approach to facilitate the biological quantification of a Nogo-A antibody using PC-12 cells as an in vitro neuronal cell model. Changes in the mRNA levels of the neuronal differentiation markers, growth-associated protein 43 and neurofilament light-polypeptide, suggest that activation of the Nogo-A pathway suppresses axonal growth and dendrite formation in the tested cell line. We found that application of anti-Nogo-A monoclonal antibody can significantly enhance the neuronal maturity of PC-12 cells by blocking the Nogo-A inhibitory effects, providing enhanced effects on neural maturity at the molecular level. No adverse effects were observed on cell viability. PMID:27027860

  10. Luteolin enhances cholinergic activities in PC12 cells through ERK1/2 and PI3K/Akt pathways.

    PubMed

    El Omri, Abdelfatteh; Han, Junkyu; Kawada, Kiyokazu; Ben Abdrabbah, Manef; Isoda, Hiroko

    2012-02-09

    Luteolin, a 3', 4', 5, 7-tetrahydroxyflavone, is an active compound in Rosmarinus officinalis (Lamiacea), and has been reported to exert several benefits in neuronal cells. However cholinergic-induced activities of luteolin still remain unknown. Neuronal differentiation encompasses an elaborate developmental program which plays a key role in the development of the nervous system. The advent of several cell lines, like PC12 cells, able to differentiate in culture proved to be the turning point for gaining and understanding of molecular neuroscience. In this work, we investigated the ability of luteolin to induce PC12 cell differentiation and its effect on cholinergic activities. Our findings showed that luteolin treatment significantly induced neurite outgrowth extension, enhanced acetylcholinesterase (AChE) activity, known as neuronal differentiation marker, and increased the level of total choline and acetylcholine in PC12 cells. In addition, luteolin persistently, activated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; while the addition of pharmacological MEK/ERK1/2 inhibitor (U0126) and PI3k/Akt inhibitor (LY294002) attenuated luteolin-induced AChE activity and neurite outgrowth in PC12 cells. The above findings suggest that luteolin induces neurite outgrowth and enhanced cholinergic activities, at least in part, through the activation of ERK1/2 and Akt signaling.

  11. Cytoprotective effects of fisetin against hypoxia-induced cell death in PC12 cells.

    PubMed

    Chen, Pei-Yi; Ho, Yi-Ru; Wu, Ming-Jiuan; Huang, Shun-Ping; Chen, Po-Kong; Tai, Mi-Hsueh; Ho, Chi-Tang; Yen, Jui-Hung

    2015-01-01

    Fisetin (3,7,3',4'-tetrahydroxyflavone), a flavonol compound of flavonoids, exhibits a broad spectrum of biological activities including anti-oxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The aim of this study is to investigate the cytoprotective effect of fisetin and the underlying molecular mechanism against hypoxia-induced cell death in PC12 cells. The results of this study showed that fisetin significantly restored the cell viability of PC12 cells under both cobalt chloride (CoCl₂)- and low oxygen-induced hypoxic conditions. Treatment with fisetin successfully reduced the CoCl₂-mediated reactive oxygen species (ROS) production, which was accompanied by an increase in the cell viability of PC12 cells. Furthermore, we found that treatment of PC12 cells with fisetin markedly upregulated hypoxia-inducible factor 1α (HIF-1α), its nuclear accumulation and the hypoxia-response element (HRE)-driven transcriptional activation. The fisetin-mediated cytoprotection during CoCl₂ exposure was significantly attenuated through the administration of HIF-1α siRNA. Moreover, we demonstrated that MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK and phosphatidylinositol 3-kinase (PI3 K) inhibitors significantly blocked the increase in cell survival that was induced by fisetin treatment under hypoxic conditions. Consistently, increased phosphorylation of ERK, p38 and Akt proteins was observed in PC12 cells treated with fisetin. However, the fisetin-induced HRE-driven transcription was not affected by inhibition of these kinase signaling pathways. Current results reveal for the first time that fisetin promotes cell survival and protects against hypoxia-induced cell death through ROS scavenging and the activation of HIF1α-, MAPK/ERK-, p38 MAPK- and PI3 K/Akt-dependent signaling pathways in PC12 cells.

  12. Protective effects of ginsenoside Rg1 against colistin sulfate-induced neurotoxicity in PC12 cells.

    PubMed

    Jiang, Guo-Zheng; Li, Ji-Chang

    2014-03-01

    The present study aimed to examine the protective effect of ginsenoside Rg1 against colistin-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Ginsenoside Rg1 was shown to elevate cell viability, decrease levels of malondialdehyde and intracellular reactive oxygen species, enhance activity of superoxide dismutase and glutathione, and decrease the release of cytochrome-c, formation of DNA fragmentation in colistin-treated PC12 cells. Ginsenoside Rg1 also reversed the increased caspase-9 and -3 mRNA levels caused by colistin in PC12 cells. These results suggest that ginsenoside Rg1 exerts a neuroprotective effect on colistin-induced neurotoxicity in PC12 cells, at least in part, via the inhibition of oxidative stress, prevention of apoptosis mediated via mitochondria pathway. Co-administration of ginsenoside Rg1 highlights the potential to increase the therapeutic index of colistin.

  13. Polysaccharides purified from Cordyceps cicadae protects PC12 cells against glutamate-induced oxidative damage.

    PubMed

    Olatunji, Opeyemi J; Feng, Yan; Olatunji, Oyenike O; Tang, Jian; Wei, Yuan; Ouyang, Zhen; Su, Zhaoliang

    2016-11-20

    Two polysaccharides CPA-1 and CPB-2 were isolated purified from Cordyceps cicadae by hot water extraction, ethanol precipitation and purification using anion exchange and gel filtration chromatography. Preliminary structural characterization of CPA-1 and CPB-2 were performed. The protective effect of CPA-1 and CPB-2 against glutamate-induced oxidative toxicity in PC12 cells was analyzed. The results indicated that pretreatment of PC12 cells with CPA-1 and CPB-2 significantly increased cell survival, Ca(2+) overload and ROS generation. CPA-1 and CPB-2 also markedly up-regulated the antioxidant status of pretreated PC12 cells. Our results suggested that Cordyceps cicadae polysaccharides can protect PC12 cells against glutamate excitotoxicity and might serve as therapeutic agents for neuronal disorders.

  14. ALTERATION OF CATECHOLAMINES IN PHOECHROMOCYTOMA (PC12) CELLS IN VITRO BY THE METABOLITES OF CHLOROTRIAZINE HERBICIDE

    EPA Science Inventory

    The effects of four major chlorotriazine metabolites on the constitutive synthesis of the catecholamines dopamine (DA) and norepinephrine (NE) were examined using undifferentiated PC12 cells. NE release and intracellular DA and NE concentrations were quantified following treatme...

  15. Capillary isoelectric focusing-tandem mass spectrometry and reversed-phase liquid chromatography-tandem mass spectrometry for quantitative proteomic analysis of differentiating PC12 cells by eight-plex isobaric tags for relative and absolute quantification.

    PubMed

    Zhu, Guijie; Sun, Liangliang; Keithley, Richard B; Dovichi, Norman J

    2013-08-06

    We report the application of capillary isoelectric focusing for quantitative analysis of a complex proteome. Biological duplicates were generated from PC12 cells at days 0, 3, 7, and 12 following treatment with nerve growth factor. These biological duplicates were digested with trypsin, labeled using eight-plex isobaric tags for relative and absolute quantification (iTRAQ) chemistry, and pooled. The pooled peptides were separated into 25 fractions using reversed-phase liquid chromatography (RPLC). Technical duplicates of each fraction were separated by capillary isoelectric focusing (cIEF) using a set of amino acids as ampholytes. The cIEF column was interfaced to an Orbitrap Velos mass spectrometer with an electrokinetically pumped sheath-flow nanospray interface. This HPLC-cIEF-electrospray-tandem mass spectrometry (ESI-MS/MS) approach identified 835 protein groups and produced 2,329 unique peptides IDs. The biological duplicates were analyzed in parallel using conventional strong-cation exchange (SCX)-RPLC-ESI-MS/MS. The iTRAQ peptides were first separated into eight fractions using SCX. Each fraction was then analyzed by RPLC-ESI-MS/MS. The SCX-RPLC approach generated 1,369 protein groups and 3,494 unique peptide IDs. For protein quantitation, 96 and 198 differentially expressed proteins were obtained with RPLC-cIEF and SCX-RPLC, respectively. The combined set identified 231 proteins. Protein expression changes measured by RPLC-cEIF and SCX-RPLC were highly correlated.

  16. Cerebrolysin protects PC12 cells from CoCl2-induced hypoxia employing GSK3β signaling.

    PubMed

    Hartwig, Kerstin; Fackler, Viktoria; Jaksch-Bogensperger, Heidi; Winter, Stefan; Furtner, Tanja; Couillard-Despres, Sebastien; Meier, Dieter; Moessler, Herbert; Aigner, Ludwig

    2014-11-01

    Cerebrolysin (EVER Neuro Pharma GmbH, Austria) is a peptidergic drug indicated for clinical use in stroke, traumatic brain injury and dementia. The therapeutic effect of Cerebrolysin is thought to ensure from its neurotrophic activity, which shares some properties with naturally occurring neurotrophic factors. However, the exact mechanism of action of Cerebrolysin is yet to be fully deciphered. This study aimed to investigate the neuroprotective effect of Cerebrolysin in a widely used in vitro model of hypoxia-induced neuronal cytotoxicity, namely cobalt chloride (CoCl2)-treatment of PC12 cells. CoCl2-cytotoxicity was indicated by a reduced cell-diameter, cell shrinkage, increased pro-apoptotic Caspase-activities and a decreased metabolic activity. Cerebrolysin maintained the cell-diameter of CoCl2-treated naïve PC12 cells, decreased the activation of Caspase 3/7 in CoCl2-stressed naïve PC12 cells and restored the cells' metabolic activity in CoCl2-impaired naïve and differentiated PC12 cells. Cerebrolysin treatment also decreased the levels of superoxide observed after exposure to CoCl2. Investigating the mechanism of action, we could demonstrate that Cerebrolysin application to CoCl2-stressed PC12 cells increased the phosphorylation of GSK3β, resulting in the inhibition of GSK3β. This might become clinically relevant for Alzheimer's disease, since GSK3β activity has been linked to the production of amyloid beta. Taken together, Cerebrolysin was found to have neuroprotective effects in CoCl2-induced cytotoxicity in PC12 cells.

  17. The Pseudophosphatase MK-STYX Induces Neurite-Like Outgrowths in PC12 Cells

    PubMed Central

    Flowers, Brittany M.; Rusnak, Lauren E.; Wong, Kristen E.; Banks, Dallas A.; Munyikwa, Michelle R.; McFarland, Alexander G.; Hinton, Shantá D.

    2014-01-01

    The rat pheochromocytoma PC12 cell line is a widely used system to study neuronal differentiation for which sustained activation of the extracellular signaling related kinase (ERK) pathway is required. Here, we investigate the function of MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] in neuronal differentiation. MK-STYX is a member of the MAPK phosphatase (MKP) family, which is generally responsible for dephosphorylating the ERKs. However, MK-STYX lacks catalytic activity due to the absence of the nucleophilic cysteine in the active site signature motif HC(X5)R that is essential for phosphatase activity. Despite being catalytically inactive, MK-STYX has been shown to play a role in important cellular pathways, including stress responses. Here we show that PC12 cells endogenously express MK-STYX. In addition, MK-STYX, but not its catalytically active mutant, induced neurite-like outgrowths in PC12 cells. Furthermore, MK-STYX dramatically increased the number of cells with neurite extensions in response to nerve growth factor (NGF), whereas the catalytically active mutant did not. MK-STYX continued to induce neurites in the presence of a MEK (MAP kinase kinase) inhibitor suggesting that MK-STYX does not act through the Ras-ERK/MAPK pathway but is involved in another pathway whose inactivation leads to neuronal differentiation. RhoA activity assays indicated that MK-STYX induced extensions through the Rho signaling pathway. MK-STYX decreased RhoA activation, whereas RhoA activation increased when MK-STYX was down-regulated. Furthermore, MK-STYX affected downstream players of RhoA such as the actin binding protein cofilin. The presence of MK-STYX decreased the phosphorylation of cofilin in non NGF stimulated cells, but increased its phosphorylation in NGF stimulated cells, whereas knocking down MK-STYX caused an opposite effect. Taken together our data suggest that MK-STYX may be a regulator of RhoA signaling, and

  18. Bcl-2 protects against FCCP-induced apoptosis and mitochondrial membrane potential depolarization in PC12 cells.

    PubMed

    Dispersyn, G; Nuydens, R; Connors, R; Borgers, M; Geerts, H

    1999-08-05

    This report addresses the relation between Bcl-2 and mitochondrial membrane potential (DeltaPsi(m)) in apoptotic cell death. Rat pheochromocytoma (PC12) cells are differentiated into neuron-like cells with nerve growth factor (NGF). It is known that Bcl-2 can attenuate apoptosis induced by deprivation of neurotrophic factor. The protective effect of Bcl-2 has been correlated with preservation of DeltaPsi(m). Protonophores, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), collapse the proton gradient across the mitochondrial inner membrane, resulting in a complete abolition of the mitochondrial membrane potential. Based on the analysis of morphology, of phosphatidylserine exposure and of nuclear fragmentation we conclude that FCCP induces apoptosis in PC12 cells, which can be prevented by overexpression of Bcl-2. To determine whether the cytoprotective effect of Bcl-2 is due to stabilization of DeltaPsi(m), we investigated the effect of Bcl-2 on changes in DeltaPsi(m), induced by FCCP in PC12 cells. We showed that treatment with FCCP induced a reduction in DeltaPsi(m), as assessed with the lipophilic cationic membrane potential-sensitive dye JC-1, and that Bcl-2 protects against FCCP-induced changes in NGF differentiated PC12 cells. Our data indicate that Bcl-2 protects against FCCP-induced cell death by stabilizing DeltaPsi(m).

  19. Protein tyrosine nitration is associated with cold- and drug-resistant microtubules in neuronal-like PC12 cells.

    PubMed

    Cappelletti, Graziella; Maggioni, Maria Grazia; Ronchi, Cristina; Maci, Rosalba; Tedeschi, Gabriella

    2006-06-19

    Among the myriad of cellular functions played by nitric oxide in the brain, there is increasing evidence that nitric oxide might be a primary player in the program of neurogenesis and neuronal differentiation. We have recently reported that tyrosine nitration of proteins is implicated in the signaling pathway triggered by nitric oxide during NGF-induced neuronal differentiation in PC12 cells. The cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and the cytoskeletal proteins alpha-tubulin and tau are two of the targets. Here, we have studied the association of nitrated proteins with the cytoskeletal fraction in differentiating PC12 cells following exposure to microtubule depolymerising treatments and found that nitration of the cytoskeleton correlates with the increased microtubule stability underlying the progression of neuronal differentiation. These results suggest a novel functional role for nitrated cytoskeletal proteins in the stabilisation of neurites occurring in differentiated neuronal cells.

  20. Changes in intracellular copper concentration and copper-regulating gene expression after PC12 differentiation into neurons

    PubMed Central

    Ogra, Yasumitsu; Tejima, Aya; Hatakeyama, Naohiro; Shiraiwa, Moeko; Wu, Siyuan; Ishikawa, Tsutomu; Yawata, Ayako; Anan, Yasumi; Suzuki, Noriyuki

    2016-01-01

    It is suspected that some neurodegenerative diseases are a result of the disturbance of copper (Cu) homeostasis, although it remains unclear whether the disturbance of Cu homeostasis has aberrant effects on neurons. Herein, we investigated Cu metabolism specifically in neurons in terms of changes in the intracellular Cu concentration and the expression of Cu-regulating genes, such as Cu transporters and metallothioneins (MTs), before and after the differentiation of rat pheochromocytoma cells (PC12 cells) into neurons. After the differentiation, Cu and Zn imaging with fluorescent probes revealed an increase in intracellular Cu concentration. The concentrations of other essential metals, which were determined by an inductively coupled plasma mass spectrometer, were not altered. The mRNA expression of the Cu influx transporter, Ctr1, was decreased after the differentiation, and the differentiated cells acquired tolerance to Cu and cisplatin, another substrate of Ctr1. In addition, the expression of MT-3, a brain-specific isoform, was increased, contrary to the decreased expression of MT-1 and MT-2. Taken together, the differentiation of PC12 cells into neurons induced MT-3 expression, thereby resulting in intracellular Cu accumulation. The decrease in Ctr1 expression was assumed to be a response aimed at abolishing the physiological accumulation of Cu after the differentiation. PMID:27623342

  1. Quantitative Assessment of Neurite Outgrowth in PC12 Cells

    EPA Science Inventory

    In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity. In order to identify potential developmental neurotoxicants, assessment of critical neurodevelopmental processes such as neuronal differenti...

  2. Tieg1/Klf10 is upregulated by NGF and attenuates cell cycle progression in the pheochromocytoma cell line PC12.

    PubMed

    Spittau, Gabriele; Happel, Nicole; Behrendt, Maik; Chao, T Ivo; Krieglstein, Kerstin; Spittau, Björn

    2010-07-01

    The transcription factor Tieg1/Klf10 belongs to a family of Sp1/Klf proteins that have been shown to play important roles during development and maintenance of various tissues and cell types. Upregulation of Tieg1/Klf10 has been reported for TGF-beta, BMP2, BMP4, ActivinA and GDNF as members of the TGF-beta superfamily. Moreover, estrogen, the cytostatic drugs homoharringtonine and velcade as well as nitric oxide are also able to trigger Tieg1/Klf10 transcription. Recent studies suggest a role for members of the neurotrophin family in regulating Tieg1/Klf10 transcriptional upregulation. Using semi-quantitative RT-PCR and immunoblotting, we present data describing that nerve growth factor (NGF) regulates the expression of Tieg1/Klf10 in the pheochromocytoma cell line PC12 in a TrkA-dependent manner. Moreover, we provide evidence for the existence of NGF-responsive elements in the 5'-regulatory region of Tieg1/Klf10 that contain binding sites for the transcription factors Sp1 and CREB. After treatment with NGF PC12 cells exit the cell cycle and start to differentiate towards a neuron-like phenotype indicated by neurite outgrowth. Using flow cytometry and differentiation assays we demonstrate that Tieg1/Klf10 reduces cell cycle progression in PC12 cells but fails to promote their terminal differentiation. Together, our results identify Tieg1/Klf10 as a new NGF target gene and substantiate its anti-proliferative function in the NGF signaling pathway in PC12 cells.

  3. Beneficial effect of astragalosides on stroke condition using PC12 cells under oxygen glucose deprivation and reperfusion.

    PubMed

    Chiu, Bi-Ying; Chang, Ching-Ping; Lin, Jia-Wei; Yu, Jung-Sheng; Liu, Wen-Pin; Hsu, Yao-Chin; Lin, Mao-Tsun

    2014-08-01

    Astragalosides (AST) are reported to be neuroprotective in focal cerebral ischemic models in vivo. In this study, the direct effect of AST against oxygen and glucose deprivation (OGD) including neuronal injury and the underlying mechanisms in vitro were investigated. 5 h OGD followed by 24 h of reperfusion [adding back oxygen and glucose (OGD-R)] was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 µg/mL) were added to the culture after 5 h of the OGD ischemic insult and was present during the reoxygenation phases. A key finding was that OGD-R decreased cell viability, increased lactate dehydrogenase, increased reactive oxygen species, apoptosis, autophagy, functional impairment of mitochondria, and endoplasmic reticulum stress in PC12 cells, all of which AST treatment significantly reduced. In addition, AST attenuated OGD-R-induced cell loss through P38 MAPK activation a neuroprotective effect blunted by SB203580, a specific inhibitor of P38 MAPK. Our data suggest that both apoptosis and autophagy are important characteristics of OGD-R-induced PC12 death and that treating PC12 cells with AST blocked OGD-R-induced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and endoplasmic reticulum stress. Our data provide identification of AST that can concomitantly inhibit multiple cells death pathways following OGD injuries in neural cells.

  4. Silymarin protects against acrylamide-induced neurotoxicity via Nrf2 signalling in PC12 cells.

    PubMed

    Li, Liang; Sun, Hong-Yang; Liu, Wei; Zhao, Hong-Yu; Shao, Mei-Li

    2017-04-01

    Silymarin (SM) is a well-known antioxidant, anti-inflammatory and anti-cancer compound extracted from the milk thistle. Here, we investigated the protective effect of SM against acrylamide (AA)-induced neurotoxicity, mainly caused by oxidative stress, via activation of the nuclear transcription factor E2-related factor 2 (Nrf2) signalling pathway in PC12 cells. The MTT reduction assay was used to measure cell viability in various drug-treated groups and demonstrated that SM could increase cell viability in AA-treated PC12 cells. We then measured the reactive oxygen species (ROS) levels by the peroxide-sensitive fluorescent probe DCFH-DA and intracellular glutathione (GSH) and malondialdehyde (MDA) levels by absorption spectrophotometry. Our data revealed that SM could reduce ROS and MDA levels and increase GSH levels in AA-induced PC12 cells. To identify a potential mechanism for SM-induced protection, we measured the mRNA and protein expression levels of Nrf2 and its downstream target antioxidants glutathione peroxidase (Gpx), glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modifier subunit (GCLM) by quantitative real-time PCR and Western blot, respectively. The results suggested that SM could activate Nrf2 signalling and increase the expression of Nrf2, Gpx, GCLC and GCLM in AA-treated PC12 cells. In conclusion, SM can effectively alleviate AA-induced neurotoxicity in PC12 cells.

  5. Daidzin protects PC12 cells from serum deprivation-induced apoptosis.

    PubMed

    Ji, Zhao-Ning; Liu, Guo-Qing

    2002-12-01

    This article examines the effect of daidzin on PC12 cell apoptosis induced by serum-free medium. PC12 cell survival was measured by MTT assay. The DNA content and percentage of apoptosis were monitored by flow cytometry and DNA fragmentation was analyzed by agarose gel electrophoresis. The results showed that serum-free (12 h) medium induced apoptosis in PC12 cells. When the cells had been treated with daidzin (0.1, 1 microM) for 12 h, the percentage of PC12 cell apoptosis was significantly decreased to 12.21 and 4.24% from 91.94% in the group with serum deprivation, and DNA fragmentation was prevented. Daidzin (0.01-10 microM) attenuated the cytotoxic effect of sodium cyanide (20 mM), glutamate (0.5 mM) and sodium nitroprusside (0.5 mM) in a manner dependent on concentration. The results suggested that daidzin prevented PC12 cell from serum free-induced apoptosis.

  6. Phase II enzyme induction by a carotenoid, lutein, in a PC12D neuronal cell line

    SciTech Connect

    Miyake, Seiji; Kobayashi, Saori; Tsubota, Kazuo; Ozawa, Yoko

    2014-04-04

    Highlights: • Lutein reduced ROS levels in a PC12D neuronal cell line. • Lutein induced mRNAs of phase II antioxidative enzymes in PC12D neuronal cells. • Lutein increased protein levels of HO-1, SOD2, and NQO-1 in PC12D neuronal cells. • Lutein had no effect on intranuclear Nrf2 levels in PC12D neuronal cells. • Lutein did not activate potential upstream Nrf2 nuclear translocation pathways. - Abstract: The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels, implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.

  7. Nerve growth factor inhibits the synthesis of a single-stranded DNA binding protein in pheochromocytoma cells (clone PC12).

    PubMed Central

    Biocca, S; Cattaneo, A; Calissano, P

    1984-01-01

    Arrest of mitosis and neurite outgrowth induced by nerve growth factor (NGF) in rat pheochromocytoma cells (clone PC12) is accompanied by a progressive inhibition of the synthesis of a protein that binds to single-stranded but not to double-stranded DNA. Time course experiments show that this inhibition is already apparent after a 2-day incubation with NGF and is maximum (85-95%) upon achievement of complete PC12 cell differentiation. Inhibition of the synthesis of this single-stranded DNA binding protein after 48 hr of incubation with NGF is potentiated by concomitant treatment of PC12 cells with antimitotic drugs acting at different levels of DNA replication. Purification on a preparative scale of this protein and analysis of its major physicochemical properties show that: (i) it constitutes 0.5% of total soluble proteins of naive PC12 cells; (ii) its molecular weight measured by NaDodSO4/PAGE is Mr 34,000 (sucrose gradient centrifugation under nondenaturing conditions yields a sedimentation coefficient s20,w of 8.1 S, indicating that the native protein is an oligomer); (iii) amino acid analysis demonstrates a preponderance of acidic over basic residues, while electrofocusing experiments show that it has an isoelectric point around 8.0; (iv) approximately 15% of the protein is phosphorylated in vivo. It is postulated that control of the synthesis of this protein is connected with activation of a differentiative program triggered by NGF in the PC12 neoplastic cell line at some step(s) of DNA activity. Images PMID:6585787

  8. Mutants of PC12 cells with altered cyclic AMP responses

    SciTech Connect

    Block, T.; Kon, C.; Breckenridge, B.M.

    1984-10-01

    PCl2 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 ..mu..m 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(BETA,..gamma..-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, N/sub s/. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PCl2 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PCl2 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.

  9. NIF (neurite-inducing factor): a novel peptide inducing neurite formation in PC12 cells.

    PubMed

    Wagner, J A

    1986-01-01

    Neurite-inducing factor (NIF) is a novel protein that has been partially purified from mouse submaxillary glands. NIF induces neurite formation in PC12 pheochromocytoma cells, and the NIF-induced neurites are indistinguishable from NGF-induced neurites in both their morphology and the time course of their formation. Neurite-inducing activity can be recovered at a position corresponding to a molecular weight of 20,000 Da after fractionation of partially purified preparations via SDS-PAGE. Partially purified preparations of NIF are about half as potent as pure beta NGF, and since the neurite-inducing activity does not correspond to any of the major proteins in this fraction, specific activity of purified NIF will probably be significantly greater than the 60 ng/ml found for our partially purified material. NIF is distinct from beta NGF by four criteria: (1) antibodies to beta NGF can block the activity of beta NGF, but not the activity of NIF; (2) beta NGF can induce ornithine decarboxylase (ODC) in PC12 cells at concentrations significantly below those required to induce neurites, while NIF induces ODC only at concentrations greatly in excess of those required to induce neurite formation; (3) by the criterion of SDS-PAGE, there is insufficient beta NGF in our partially purified preparations of NIF to explain the biological activity of this fraction; and (4) the biological activity of NIF has a molecular weight (20,000 Da) that is distinct from beta NGF (13,000 Da). We conclude that NIF is probably a novel peptide that is very active in promoting morphological differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. SOD2 Mediates Amifostine-Induced Protection against Glutamate in PC12 Cells

    PubMed Central

    Jia, Ji; Zhang, Lei; Shi, Xiaolei; Wu, Mingchun; Zhou, Xiang; Liu, Xiaonan; Huo, Tingting

    2016-01-01

    Background. Cytoprotectant amifostine attenuates radiation-induced oxidative injury by increasing intracellular manganese superoxide dismutase (SOD2) in peripheral tissue. However, whether amifostine could protect neuronal cells against oxidative injury has not been reported. The purpose of this study is to explore the protection of amifostine in PC12 cells. Methods. PC12 cells exposed to glutamate were used to mimic neuronal oxidative injury. SOD assay kit was taken to evaluate intracellular Cu/Zn SOD (SOD1) and SOD2 activities; western blot analysis and immunofluorescence staining were performed to investigate SOD2 protein expression; MTT, lactate dehydrogenase (LDH), release and cell morphology were used to evaluate cell injury degree, and apoptotic rate and cleaved caspase-3 expression were taken to assess apoptosis; mitochondrial superoxide production, intracellular reactive oxygen species (ROS), and glutathione (GSH) and catalase (CAT) levels were evaluated by reagent kits. Results. Amifostine increased SOD2 activity and expression, decreased cell injury and apoptosis, reduced mitochondrial superoxide production and intracellular ROS generation, and restored intracellular GSH and CAT levels in PC12 cells exposed to glutamate. SOD2-siRNA, however, significantly reversed the amifostine-induced cytoprotective and antioxidative actions. Conclusion. SOD2 mediates amifostine-induced protection in PC12 cells exposed to glutamate. PMID:26770652

  11. Chloride channels involve in hydrogen peroxide-induced apoptosis of PC12 cells.

    PubMed

    Zuo, Wanhong; Zhu, Linyan; Bai, Zhiquan; Zhang, Haifeng; Mao, Jianwen; Chen, Lixin; Wang, Liwei

    2009-10-02

    Chloride channel activity is one of the critical factors responsible for cell apoptotic volume decrease (AVD). However, the roles of chloride channels in apoptosis have not been fully understood. In the current study, we assessed the role of chloride channels in hydrogen peroxide (H(2)O(2))-induced apoptosis of pheochromocytoma cells (PC12). Extracellular application of H(2)O(2) activated a chloride current and induced cell volume decrease in a few minutes. Incubation of cells with H(2)O(2) elevated significantly the membrane permeability to the DNA dye Hoechst 33258 in 1h and induced apoptosis of most PC12 cells tested in 24h. The chloride channel blocker NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) prevented appearance of H(2)O(2)-induced high membrane permeability and cell shrinkage, suppressed H(2)O(2)-activated chloride currents and protected PC12 cells from apoptosis induced by H(2)O(2). The results suggest that chloride channels may contribute to H(2)O(2)-induced apoptosis by ways of elevation of membrane permeability and AVD in PC12 cells.

  12. Enhanced proliferation of PC12 neural cells on untreated, nanotextured glass coverslips

    NASA Astrophysics Data System (ADS)

    Islam, Muhymin; Atmaramani, Rahul; Mukherjee, Siddhartha; Ghosh, Santaneel; Iqbal, Samir M.

    2016-10-01

    Traumatic injury to the central nervous system is a significant health problem. There is no effective treatment available partly because of the complexity of the system. Implementation of multifunctional micro- and nano-device based combinatorial therapeutics can provide biocompatible and tunable approaches to perform on-demand release of specific drugs. This can help the damaged cells to improve neuronal survival, regeneration of axons, and their reconnection to appropriate targets. Nano-topological features induced rapid cell growth is especially important towards the design of effective platforms to facilitate damaged neural circuit reconstruction. In this study, for the first time, feasibility of neuron-like PC12 cell growth on untreated and easy to prepare nanotextured surfaces has been carried out. The PC12 neuron-like cells were cultured on micro reactive ion etched nanotextured glass coverslips. The effect of nanotextured topology as physical cue for the growth of PC12 cells was observed exclusively, eliminating the possible influence(s) of the enhanced concentration of coated materials on the surface. The cell density was observed to increase by almost 200% on nanotextured coverslips compared to plain coverslips. The morphology study indicated that PC12 cell attachment and growth on the nanotextured substrates did not launch any apoptotic machinery of the cell. Less than 5% cells deformed and depicted condensed nuclei with apoptotic bodies on nanotextured surfaces which is typical for the normal cell handling and culture. Enhanced PC12 cell proliferation by such novel and easy to prepare substrates is not only attractive for neurite outgrowth and guidance, but may be used to increase the affinity of similar cancerous cells (ex: B35 neuroblastoma) and rapid proliferation thereafter—towards the development of combinatorial theranostics to diagnose and treat aggressive cancers like neuroblastoma.

  13. Enhanced proliferation of PC12 neural cells on untreated, nanotextured glass coverslips.

    PubMed

    Islam, Muhymin; Atmaramani, Rahul; Mukherjee, Siddhartha; Ghosh, Santaneel; Iqbal, Samir M

    2016-10-14

    Traumatic injury to the central nervous system is a significant health problem. There is no effective treatment available partly because of the complexity of the system. Implementation of multifunctional micro- and nano-device based combinatorial therapeutics can provide biocompatible and tunable approaches to perform on-demand release of specific drugs. This can help the damaged cells to improve neuronal survival, regeneration of axons, and their reconnection to appropriate targets. Nano-topological features induced rapid cell growth is especially important towards the design of effective platforms to facilitate damaged neural circuit reconstruction. In this study, for the first time, feasibility of neuron-like PC12 cell growth on untreated and easy to prepare nanotextured surfaces has been carried out. The PC12 neuron-like cells were cultured on micro reactive ion etched  nanotextured glass coverslips. The effect of nanotextured topology as physical cue for the growth of PC12 cells was observed exclusively, eliminating the possible influence(s) of the enhanced concentration of coated materials on the surface. The cell density was observed to increase by almost 200% on nanotextured coverslips compared to plain coverslips. The morphology study indicated that PC12 cell attachment and growth on the nanotextured substrates did not launch any apoptotic machinery of the cell. Less than 5% cells deformed and depicted condensed nuclei with apoptotic bodies on nanotextured surfaces which is typical for the normal cell handling and culture. Enhanced PC12 cell proliferation by such novel and easy to prepare substrates is not only attractive for neurite outgrowth and guidance, but may be used to increase the affinity of similar cancerous cells (ex: B35 neuroblastoma) and rapid proliferation thereafter-towards the development of combinatorial theranostics to diagnose and treat aggressive cancers like neuroblastoma.

  14. Neurological morphofunctional differentiation induced by REAC technology in PC12. A neuro protective model for Parkinson's disease.

    PubMed

    Maioli, Margherita; Rinaldi, Salvatore; Migheli, Rossana; Pigliaru, Gianfranco; Rocchitta, Gaia; Santaniello, Sara; Basoli, Valentina; Castagna, Alessandro; Fontani, Vania; Ventura, Carlo; Serra, Pier Andrea

    2015-05-15

    Research for the use of physical means, in order to induce cell differentiation for new therapeutic strategies, is one of the most interesting challenges in the field of regenerative medicine, and then in the treatment of neurodegenerative diseases, Parkinson's disease (PD) included. The aim of this work is to verify the effect of the radio electric asymmetric conveyer (REAC) technology on the PC12 rat adrenal pheochromocytoma cell line, as they display metabolic features of PD. PC12 cells were cultured with a REAC regenerative tissue optimization treatment (TO-RGN) for a period ranging between 24 and 192 hours. Gene expression analysis of specific neurogenic genes, as neurogenin-1, beta3-tubulin and Nerve growth factor, together with the immunostaining analysis of the specific neuronal protein beta3-tubulin and tyrosine hydroxylase, shows that the number of cells committed toward the neurogenic phenotype was significantly higher in REAC treated cultures, as compared to control untreated cells. Moreover, MTT and Trypan blue proliferation assays highlighted that cell proliferation was significantly reduced in REAC TO-RGN treated cells. These results open new perspectives in neurodegenerative diseases treatment, particularly in PD. Further studies will be needed to better address the therapeutic potential of the REAC technology.

  15. Comparative toxicity and apoptosis induced by diorganotins in rat pheochromocytoma (PC12) cells.

    PubMed

    Liu, Enli; Du, Xue; Ge, Rui; Liang, Taigang; Niu, Qiao; Li, Qingshan

    2013-10-01

    As ubiquitous environmental toxicants, organotin (IV) compounds (OTC) accumulate in the food chain and potential effects on human health are disquieting. The present study compared the cytotoxicity of three diorganotins, namely, dimethyltin (DMT), dibutyltin (DBT) and diphenyltin (DPT), in rat pheochromocytoma (PC12) cells, and the molecular mechanisms responsible for their cytotoxic effects were also explored. Twenty-four hours exposure of PC12 cells to DBT and DPT resulted in a concentration-dependent decrease in cell viability with median lethal concentration (LC₅₀) of 2.97 μM and 7.24 μM, respectively. However, DMT at concentrations up to 128 μM had no obvious effect on cell viability. The mechanistic study revealed that the extent of apoptosis was greater for DBT than that for DPT, followed by DMT, as evidenced by acridine orange/ethidium bromide (AO/EB) fluorescent staining method and annexin V-FITC/PI staining flow cytometry analysis, as well as generation of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) disruption, release of cytochrome c (Cyt c), and consequent activation of caspase-9, and -3. These investigations suggested that the cytotoxic potency of three diorganotins in PC12 cells was in the order of DBT>DPT≫DMT, and these compounds could induce PC12 cells apoptosis through ROS mediated mitochondrial pathway.

  16. Cytotoxic, Genotoxic, and Neurotoxic Effects of Mg, Pb, and Fe on Pheochromocytoma (PC-12) Cells

    PubMed Central

    Sanders, Talia; Liu, Yi-Ming; Tchounwou, Paul B.

    2014-01-01

    Metals such as lead (Pb), magnesium (Mg), and iron (Fe) are ubiquitous in the environment as a result of natural occurrence and anthropogenic activities. Although Mg, Fe and others are considered essential elements, high level of exposure has been associated with severe adverse health effects including cardiovascular, hematological, nephrotoxic, hepatotoxic, and neurologic abnormalities in humans. In the present study we hypothesized that Mg, Pb, and Fe are cytotoxic, genotoxic and neurotoxic, and their toxicity is mediated through oxidative stress and alteration in protein expression. To test the hypothesis, we used the pheochromocytoma (PC-12) cell line as a neuro cell model and performed the LDH assay for cell viability, Comet assay for DNA damage, Western blot for oxidative stress, and HPLC-MS to assess the concentration levels of neurological biomarkers such as glutamate, dopamine (DA), and 3-methoxytyramine (3-MT). The results of this study clearly show that Mg, Pb, and Fe, respectively in the form of MgSO4, Pb(NO3)2, FeCl2, and FeCl3 induce cytotoxicity, oxidative stress, and genotoxicity in PC-12 cells. In addition, exposure to these metallic compounds caused significant changes in the concentration levels of glutamate, dopamine, and 3-MT in PC-12 cells. Taken together the findings suggest that MgSO4, Pb(NO3)2, FeCl2, and FeCl3 have the potential to induce substantial toxicity to PC-12 cells. PMID:24942330

  17. Effects of 60-GHz millimeter waves on neurite outgrowth in PC12 cells using high-content screening.

    PubMed

    Haas, Alexis J; Le Page, Yann; Zhadobov, Maxim; Sauleau, Ronan; Le Dréan, Yves

    2016-04-08

    Technologies for wireless telecommunication systems using millimeter waves (MMW) will be widely deployed in the near future. Forthcoming applications in this band, especially around 60GHz, are mainly developed for high data-rate local and body-centric telecommunications. At those frequencies, electromagnetic radiations have a very shallow penetration into biological tissues, making skin keratinocytes, and free nerve endings of the upper dermis the main targets of MMW. Only a few studies assessed the impact of MMW on neuronal cells, and none of them investigated a possible effect on neuronal differentiation. We used a neuron-like cell line (PC12), which undergoes neuronal differentiation when treated with the neuronal growth factor (NGF). PC12 cells were exposed at 60.4GHz for 24h, at an incident power density averaged over the cell monolayer of 10mW/cm(2). Using a large scale cell-by-cell analysis based on high-content screening microscopy approach, we assessed potential effects of MMW on PC12 neurite outgrowth and cytoskeleton protein expression. No differences were found in protein expression of the neuronal marker β3-tubulin nor in internal expression control β-tubulin. On the other hand, our data showed a slight increase, although insignificant, in neurite outgrowth, induced by MMW exposure. However, experimental controls demonstrated that this increase was related to heating.

  18. Deoxynivalenol induces apoptosis in PC12 cells via the mitochondrial pathway.

    PubMed

    Wang, Xichun; Xu, Wei; Fan, Mengxue; Meng, Tingting; Chen, Xiaofang; Jiang, Yunjing; Zhu, Dianfeng; Hu, Wenjuan; Gong, Jiajie; Feng, Shibin; Wu, Jinjie; Li, Yu

    2016-04-01

    Deoxynivalenol (DON) has broad toxicity in animals and humans. In this study the impact of DON treatment on apoptotic pathways in PC12 cells was determined. The effects of DON were evaluated on (i) typical indicators of apoptosis, including cellular morphology, cell activity, lactate dehydrogenase (LDH) release, and apoptosis ratio in PC12 cells, and on (ii) the expression of key genes and proteins related to apoptosis, including Bcl-2, Bax, Bid, cytochrome C (Cyt C), apoptosis inducing factor (AIF), cleaved-Caspase9, and cleaved-Caspase3. DON treatment inhibited proliferation of PC12 cells, induced significant morphological changes and apoptosis, promoted the release of Cyt C and AIF from the mitochondria, and increased the activities of cleaved-Caspase9 and cleaved-Caspase3. Bcl-2 expression decreased with increasing DON concentrations, in contrast to Bax and Bid, which were increased with increasing DON concentration. These data demonstrate that DON induces apoptosis in PC12 cells through the mitochondrial apoptosis pathway.

  19. POTENTIAL MECHANISMS RESPONSIBLE FOR CHLOROTRIAZINE-INDUCED ALTERATIONS IN CATECHOLAMINES IN PHEOCHROMOCYTOMA (PC12) CELLS

    EPA Science Inventory

    ABSTRACT

    Potential Mechanisms Responsible for Chlorotriazine-induced Changes in Catecholamine Metabolism in Pheochromocytoma (PC12) Cells*
    PARIKSHIT C. DAS1, WILLIAM K. McELROY2 , AND RALPH L. COOPER2+
    1Curriculum in Toxicology, University of North Carolina, Chape...

  20. Nerve growth factor withdrawal-induced cell death in neuronal PC12 cells resembles that in sympathetic neurons

    PubMed Central

    1992-01-01

    Previous studies have shown that in neuronal cells the developmental phenomenon of programmed cell death is an active process, requiring synthesis of both RNA and protein. This presumably reflects a requirement for novel gene products to effect cell death. It is shown here that the death of nerve growth factor-deprived neuronal PC12 cells occurs at the same rate as that of rat sympathetic neurons and, like rat sympathetic neurons, involves new transcription and translation. In nerve growth factor-deprived neuronal PC12 cells, a decline in metabolic activity, assessed by uptake of [3H]2-deoxyglucose, precedes the decline in cell number, assessed by counts of trypan blue-excluding cells. Both declines are prevented by actinomycin D and anisomycin. In contrast, the death of nonneuronal (chromaffin-like) PC12 cells is not inhibited by transcription or translation inhibitors and thus does not require new protein synthesis. DNA fragmentation by internucleosomal cleavage does not appear to be a consistent or significant aspect of cell death in sympathetic neurons, neuronal PC12 cells, or nonneuronal PC12 cells, notwithstanding that the putative nuclease inhibitor aurintricarboxylic acid protects sympathetic neurons, as well as neuronal and nonneuronal PC12 cells, from death induced by trophic factor removal. Both phenotypic classes of PC12 cells respond to aurintricarboxylic acid with similar dose-response characteristics. Our results indicate that programmed cell death in neuronal PC12 cells, but not in nonneuronal PC12 cells, resembles programmed cell death in sympathetic neurons in significant mechanistic aspects: time course, role of new protein synthesis, and lack of a significant degree of DNA fragmentation. PMID:1469055

  1. Effects of Salidroside on cobalt chloride-induced hypoxia damage and mTOR signaling repression in PC12 cells.

    PubMed

    Zhong, Xiaoyong; Lin, Ruhui; Li, Zuanfang; Mao, Jingjie; Chen, Lidian

    2014-01-01

    Salidroside (SA), a phenylpropanoid glycoside isolated from Rhodiola rosea L., has been documented to exert a broad spectrum of pharmacological properties, including protective effects against neuronal death induced by various stresses. To provide further insights into the neuroprotective functions of SA, this study examined whether SA can attenuate cobalt chloride (CoCl2)-induced hypoxia damage and mammalian target of rapamycin (mTOR) signaling repression in PC12 differentiated cells. Differentiated PC12 cells were exposed to CoCl2 for 12 h to mimic hypoxic/ischemic conditions and treated with SA at the same time, followed by electron microscopy and analysis of cell viability, intracellular reactive oxygen species (ROS) level, hypoxia-inducible factor-1α (HIF-1α) level, and the regulated in development and DNA damage responses (REDD1)/mTOR/ p70 ribosomal S6 kinase (p70S6K) signaling pathway. Our data indicated that SA can dramatically attenuate the ultrastructural damage of mitochondria induced by CoCl2 and significantly decrease CoCl2-induced ROS production. Moreover, phosphorylated mammalian target of rapamycin (p-mTOR) was significantly reduced by CoCl2, and this inhibition was relieved by the treatment of SA in PC12 cells, as evidenced by immunoblot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The SA effects were blocked by pretreatment of RAD001. The results indicate that SA can rescue CoCl2-induced repression of REDD1/mTOR/ p70S6K signal transduction in PC12 cells. Our data demonstrate that SA is able to attenuate CoCl2-induced hypoxia damage and mTOR signaling repression, suggesting that SA may protect brain neurons from ischemic injury through mTOR signaling, and provide new insights into the prevention and treatment of cerebral ischemic.

  2. Rab22 controls NGF signaling and neurite outgrowth in PC12 cells.

    PubMed

    Wang, Liang; Liang, Zhimin; Li, Guangpu

    2011-10-01

    Rab22 is a small GTPase that is localized on early endosomes and regulates early endosomal sorting. This study reports that Rab22 promotes nerve growth factor (NGF) signaling-dependent neurite outgrowth and gene expression in PC12 cells by sorting NGF and the activated/phosphorylated receptor (pTrkA) into signaling endosomes to sustain signal transduction in the cell. NGF binding induces the endocytosis of pTrkA into Rab22-containing endosomes. Knockdown of Rab22 via small hairpin RNA (shRNA) blocks NGF-induced pTrkA endocytosis into the endosomes and gene expression (VGF) and neurite outgrowth. Overexpression of human Rab22 can rescue the inhibitory effects of the Rab22 shRNA, suggesting a specific Rab22 function in NGF signal transduction, rather than off-target effects. Furthermore, the Rab22 effector, Rabex-5, is necessary for NGF-induced neurite outgrowth and gene expression, as evidenced by the inhibitory effect of shRNA-mediated knockdown of Rabex-5. Disruption of the Rab22-Rabex-5 interaction via overexpression of the Rab22-binding domain of Rabex-5 in the cell also blocks NGF-induced neurite outgrowth, suggesting a critical role of Rab22-Rabex-5 interaction in the biogenesis of NGF-signaling endosomes to sustain the signal for neurite outgrowth. These data provide the first evidence for an early endosomal Rab GTPase as a positive regulator of NGF signal transduction and cell differentiation.

  3. Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells

    PubMed Central

    Wang, X; Gerdes, H-H

    2015-01-01

    Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context. PMID:25571977

  4. Dp71, utrophin and beta-dystroglycan expression and distribution in PC12/L6 cell cocultures

    PubMed Central

    Ilarraza-Lomeli, Ramses; Cisneros-Vega, Bulmaro; Romo-Yañez, Jose; Cervantes-Gomez, Maria De Lourdes; Mornet, Dominique; Montañez, Cecilia

    2007-01-01

    Dystrophin Dp71 is the most ubiquitous and highest expressed dystrophin isoform in brain, however, Dp71 function and those specific for its spliced d- and ab- isoforms remains undetermined. To study Dp71, utrophin and β-dystroglycan in cell-to-cell interactions, we first established a co-culture model using PC 12 cells and L6 myotubes. Confocal microscopy assays of these co-cultures, in which PC 12 cells are differentiated in the presence of L6 myotubes, showed that the Dp71d isoform accumulates in PC 12 nuclei, Golgi-complex- and endoplasmic reticulum-like structures, being depleted from neurites and cytoplasm, while Dp71ab concentrates at neurite tips and cytoplasm and colocalizes with β-dystroglycan, utrophin, synaptophysin and acetylcholine receptors. Evidences suggest Dp71ab isoform unlike Dp71d, may take part in neurite-related processes. This is the first work on the role of dystrophins as well as members of the DAP complex in a cell-line based co-culturing system, which may prove useful in determining protein associations in a more controlled environment than ex-vivo systems. PMID:17921863

  5. Oligomeric Forms of Insulin Amyloid Aggregation Disrupt Outgrowth and Complexity of Neuron-Like PC12 Cells

    PubMed Central

    Kachooei, Ehsan; Moosavi-Movahedi, Ali Akbar; Khodagholi, Fariba; Ramshini, Hassan; Shaerzadeh, Fatemeh; Sheibani, Nader

    2012-01-01

    Formation of protein amyloid fibrils consists of a series of intermediates including oligomeric aggregates, proto-fibrillar structures, and finally mature fibrils. Recent studies show higher toxicity for oligomeric and proto-fibrillar intermediates of protein relative to their mature fibrils. Here the kinetic of the insulin amyloid fibrillation was evaluated using a variety of techniques including ThT fluorescence, Congo red absorbance, circular dichroism, and atomic force microscopy (AFM). The solution surface tension changes were attributed to hydrophobic changes in insulin structure and were detected by Du Noüy Ring method. Determination of the surface tension of insulin oligomeric, proto-fibrillar and fibrillar forms indicated that the hydrophobicity of solution is enhanced by the formation of the oligomeric forms of insulin compared to other forms. In order to investigate the toxicity of the different forms of insulin we monitored morphological alterations of the differentiated neuron-like PC12 cells following incubation with native, oligomeric aggregates, proto-fibrillar, and fibrillar forms of insulin. The cell body area, average neurite length, neurite width, number of primary neurites, and percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different forms of insulin. We observed that the oligomeric form of insulin impaired the growth and complexity of PC12 cells compared to other forms. Together our data suggest that the lower surface tension of oligomers and their perturbation affects the morphology of PC12 cells, mainly due to their enhanced hydrophobicity and detergent-like structures. PMID:22848469

  6. Protective effect of arctigenin on ethanol-induced neurotoxicity in PC12 cells.

    PubMed

    Huang, Jia; Xiao, Lan; Wei, Jing-Xiang; Shu, Ya-Hai; Fang, Shi-Qi; Wang, Yong-Tang; Lu, Xiu-Min

    2017-04-01

    As a neurotropic substance, ethanol can damage nerve cells through an increase in the production of free radicals, interference of neurotrophic factor signaling pathways, activation of endogenous apoptotic signals and other molecular mechanisms. Previous studies have revealed that a number of natural drugs extracted from plants offer protection of nerve cells from damage. Among these, arctigenin (ATG) is a lignine extracted from Arctium lappa (L.), which has been found to exert a neuroprotective effect on scopolamine‑induced memory deficits in mice with Alzheimer's disease and glutamate-induced neurotoxicity in primary neurons. As a result, it may offer beneficial effects on ethanol-induced neurotoxicity. However, the effects of ATG on ethanol‑induced nerve damage remain to be elucidated. To address this issue, the present study used rat pheochromocytoma PC12 cells to investigate the neuroprotective effects of ATG on ethanol-induced cell damage by performing an MTT reduction assay, cell cycle analysis, Hoechst33342/propidium iodide fluorescence staining and flow cytometry to examine apoptosis. The results showed that 10 µM ATG effectively promoted the proliferation of damaged cells, and increased the distribution ratio of the cells at the G2/M and S phases (P<0.05). In addition, the apoptosis and necrosis of the PC12 cells were significantly decreased following treatment with ATG. Therefore, it was concluded that 10 µM ATG had a protective effect on ethanol‑induced injury in PC12 cells.

  7. IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells.

    PubMed

    Nakatani, Yoshihiko; Nagaoka, Takumi; Hotta, Sayako; Utsunomiya, Iku; Yoshino, Hiide; Miyatake, Tadashi; Hoshi, Keiko; Taguchi, Kyoji

    2007-03-01

    We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0+/-9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3-4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6+/-2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1+/-9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

  8. Low Doses of Camptothecin Induced Hormetic and Neuroprotective Effects in PC12 Cells

    PubMed Central

    Zhang, Chao; Chen, Shenghui; Bao, Jiaolin; Zhang, Yulin; Huang, Borong; Jia, Xuejing; Chen, Meiwan; Wan, Jian-Bo; Su, Huanxing; Wang, Yitao

    2015-01-01

    Hormetic response is an adaptive mechanism for a cell or organism surviving in an unfavorable environment. It has been an intriguing subject of researches covering a broad range of biological and medical disciplines, in which the underlying significance and molecular mechanisms are under intensive investigation. In the present study, we demonstrated that topoisomerase I inhibitor camptothecin (CPT), a potent anticancer agent, induced an obvious hormetic response in rat pheochromocytoma PC12 cells. Camptothecin inhibited PC12 cell growth at relative high doses as generally acknowledged while stimulated the cell growth by as much as 39% at low doses. Moreover, low doses of CPT protected the cells from hydrogen peroxide (H2O2)-induced cell death. Phosphoinositide 3-kinase (PI3K)/Akt and nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathways were reported playing pivotal roles in protecting cells from oxidative stress. We observed that these 2 pathways were upregulated by low doses of CPT, as evidenced by increased levels of phosphorylated PI3K, phosphorylated Akt, phosphorylated mammalian target of rapamycin, Nrf2, and HO-1; and abolishment of the growth-promoting and neuroprotective effects of CPT by LY294002, a PI3K inhibitor. These results suggest that the hormetic and neuroprotective effects of CPT at low doses on PC12 cells were attributable, at least partially, to upregulated PI3K/Akt and Nrf2/HO-1 pathways. PMID:26674066

  9. Hydrogen gas alleviates oxygen toxicity by reducing hydroxyl radical levels in PC12 cells

    PubMed Central

    Yu, Junchao; Yu, Qiuhong; Liu, Yaling; Zhang, Ruiyun; Xue, Lianbi

    2017-01-01

    Hyperbaric oxygen (HBO) therapy through breathing oxygen at the pressure of above 1 atmosphere absolute (ATA) is useful for varieties of clinical conditions, especially hypoxic-ischemic diseases. Because of generation of reactive oxygen species (ROS), breathing oxygen gas at high pressures can cause oxygen toxicity in the central nervous system, leading to multiple neurological dysfunction, which limits the use of HBO therapy. Studies have shown that Hydrogen gas (H2) can diminish oxidative stress and effectively reduce active ROS associated with diseases. However, the effect of H2 on ROS generated from HBO therapy remains unclear. In this study, we investigated the effect of H2 on ROS during HBO therapy using PC12 cells. PC12 cells cultured in medium were exposed to oxygen gas or mixed oxygen gas and H2 at 1 ATA or 5 ATA. Cells viability and oxidation products and ROS were determined. The data showed that H2 promoted the cell viability and inhibited the damage in the cell and mitochondria membrane, reduced the levels of lipid peroxidation and DNA oxidation, and selectively decreased the levels of •OH but not disturbing the levels of O2•-, H2O2, or NO• in PC12 cells during HBO therapy. These results indicated that H2 effectively reduced •OH, protected cells against oxygen toxicity resulting from HBO therapy, and had no effect on other ROS. Our data supported that H2 could be potentially used as an antioxidant during HBO therapy. PMID:28362819

  10. Nitric oxide promotes nicotine-triggered ERK signaling via redox reactions in PC12 cells.

    PubMed

    Miyamoto, Yoshiaki; Sakai, Ryosuke; Maeda, Chiharu; Takata, Tsuyoshi; Ihara, Hideshi; Tsuchiya, Yukihiro; Watanabe, Yasuo

    2011-10-30

    Nitric oxide (NO), produced by neuronal NO synthase (nNOS), serves as a signaling molecule with diverse biological responses in the central nervous system (CNS). In the present study, we demonstrated that nNOS expression enhances the nicotine-triggered activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in nNOS-transfected PC12 (NPC12) cells. Treatment with nicotine increased the phosphorylation level of ERK1/2 in the NPC12 cells as compared with that in control PC12 cells. However, nicotine treatment failed to enhance ERK1/2 phosphorylation when NPC12 cells were pretreated with several selective inhibitors of NOS, the nicotinic acetylcholine receptors, L-type voltage-dependent Ca(2+) channels, protein kinase C, Src, epidermal growth factor receptor, and MEK. The nicotine-induced ERK1/2 phosphorylation in PC12 cells was observed by their pretreatment with a NO donor. Moreover, the enhancement of nicotine-induced ERK1/2 phosphorylation in the NPC12 cells was regulated by intracellular glutathione levels, but not by the soluble guanylate cyclase-cGMP-protein kinase G signaling. Meanwhile, depolarization stimulated ERK1/2 phosphorylation in both PC12 and NPC12 cells. Taken together, these findings suggest that nicotine modulates NO-dependent redox condition; the resulting calcium influx, would increase ERK1/2 phosphorylation in nNOS expressing cells. Blockade of NO pathway may be selective target to reduce ERK1/2 phosphorylation via attenuation of the nicotine responses in the CNS.

  11. Oxidative stress-mediated cytotoxicity of zirconia nanoparticles on PC12 and N2a cells

    NASA Astrophysics Data System (ADS)

    Asadpour, Elham; Sadeghnia, Hamid R.; Ghorbani, Ahmad; Sedaghat, Mehran; Boroushaki, Mohammad T.

    2016-01-01

    In recent years, there is a growing interest in the application of nanoparticles like zirconium dioxide (zirconia <100 nm), for many purposes. Since a comprehensive study on the toxic effects of zirconia has not been done, we decided to investigate the effects of zirconia nanoparticles on cultured PC12 and N2a cells. In this study, cytotoxic effect of different concentrations of zirconia nanoparticles at three different time intervals were evaluated using MTT and ROS (reactive oxygen species) assays. Also, Lipid peroxidation, glutathione (GSH) content changes, and DNA damage were measured. Zirconia nanoparticles caused a significant reduction in cell viability and GSH content of the cells, and induce a significant increase in intracellular ROS and MDA content of PC12 and N2a cells. Moreover, it increases the percentage of DNA tail of treated cells as compared with control group. Zirconia nanoparticles have cytotoxic and genotoxic effects in PC12 and N2a cells in a time and concentration-dependent manner in concentration more than 31 µg/mL.

  12. The metalloproteinase stromelysin-1 (transin) mediates PC12 cell growth cone invasiveness through basal laminae.

    PubMed

    Nordstrom, L A; Lochner, J; Yeung, W; Ciment, G

    1995-02-01

    Matrix metalloproteinases have been implicated in various extracellular matrix remodeling events that occur during normal development and in a number of pathologies. In previous work with PC12 rat pheochromocytoma cells, we found that the matrix metalloproteinase stromelysin-1 (ST1) was highly induced by nerve growth factor (NGF), but not by epidermal growth factor (EGF). Here, we show that ST1 immunoreactivity is present in growth cones of NGF-treated PC12 cells, but not EGF-treated or untreated cells. To determine whether ST1 expression confers neurite invasiveness, three lines of PC12 cells were produced that constitutively express ST1 antisense mRNA. These lines expressed and secreted significantly reduced levels of ST1 protein, as determined by immunoblot and immunocytochemical methods, but otherwise responded normally to NGF-treatment by elaborating neurites. We found, however, that the neurites of these ST1 antisense cells showed a significantly reduced ability to penetrate a Matrigel reconstituted basal lamina, as compared to the parental cells, suggesting that ST1 confers neurite invasiveness. Finally, we show that ST1 is also expressed in vivo in sections through Embryonic Day 15 rat embryos, including neurons of both the peripheral and central nervous systems. These data indicate that ST1 may play a role in axonal growth in vivo, including a role in growth cone invasiveness.

  13. Phosphatidylinositol-anchored glycoproteins of PC12 pheochromocytoma cells and brain

    SciTech Connect

    Margolis, R.K.; Goossen, B.; Margolis, R.U.

    1988-05-03

    PC12 pheochromocytoma cells and cultures of early postnatal rat cerebellium were labeled with (/sup 3/H)glucosamine, (/sup 3/H)fucose, (/sup 3/H)leucine, (/sup 3/H)ethanolamine, or sodium (/sup 35/S)sulfate and treated with a phosphatidylinositol-specific phospholipase C. Enzyme treatment of (/sup 3/H) glucosamine- or (/sup 3/H)fucose-labeled PC12 cells led to a 15-fold increase in released glycoproteins. On sodium dodecyl sulfate-polyacrylamide gel ectrophoresis, most of the released material migrated as a broad band with an apparent molecular size of 32,000 daltons (Da), which was specifically immunoprecipitated by a monoclonal antibody to the Thy-l glycoprotein. A second glycoprotein, with an apparent molecular size of 158,000 Da, was also released. After treatment with endo-..beta..-galactosidase, 40-45% of the (/sup 3/H)glucosamine of (/sup 3/H)fucose radioactivity in the phospholipase-released glycoproteins was converted to products of disaccharide size, and the molecular size of the 158-kDa glycoprotein decreased to 145 kDa, demonstrating that it contains fucosylated poly-(N-acetyllactosaminyl) oligosaccharides. The phospholipase also released labeled Thy-1 and the 158-kDa glycoprotein from PC12 cells cultured in the presence of (/sup 3/H)ethanolamine, which specifically labels this component of the phosphatidylinositol membrane-anchoring sequence,while in the lipid-free protein residue of cells not treated with phospholipase, Thy-1 and a doublet at 46/48 kDa were the only labeled proteins. Sulfated glycoproteins of 155, 132/134, 61, and 21 kDa are the predominant species released by phospholipase, which does not affect a major 44-kDa protein seen in (/sup 3/H)ethanolamine-labeled brain cultures. The 44-48- and 155/158-kDa proteins may be common to both PC12 cells and brain.

  14. Neuroprotective effects of constituents of Eragrostis ferruginea against Aβ-induced toxicity in PC12 cells.

    PubMed

    Na, Chae Sun; Hong, Seong Su; Choi, Yun-Hyeok; Lee, Yong Ho; Hong, Sun Hee; Lim, Ji-Youn; Kang, Byeong Hoa; Park, So-Young; Lee, Dongho

    2010-07-01

    A new flavonoid, 7-demethylageconyflavone A (1), and five known compounds, tricin (2), ageconyflavone A (3), corylin (4), nectandrin B (5), and 4-ketopinoresinol (6) were isolated from the aerial parts of Eragrostis ferruginea. Their structures were determined using spectroscopic techniques, including 1D- and 2D-NMR. All compounds were tested for the neuroprotective effects against amyloid beta peptide (Abeta) using PC12 cells, a major cause of the pathology of Alzheimer's disease. Tricin (2) was found to have a neuroprotective effect with an ED(50) value of 20.3 microM against Abeta-induced toxicity in PC12 cells. Ageconyflavone A (3), nectandrin B (5) and 4-ketopinoresinol (6) demonstrated moderate neuroprotective effects with ED(50) values of 58.7, 44.1, and 54.8 microM, respectively.

  15. Three-dimensional tracking of single secretory granules in live PC12 cells.

    PubMed

    Li, Dongdong; Xiong, Jun; Qu, Anlian; Xu, Tao

    2004-09-01

    Deconvolution wide-field fluorescence microscopy and single-particle tracking were used to study the three-dimensional mobility of single secretory granules in live PC12 cells. Acridine orange-labeled granules were found to travel primarily in random and caged diffusion, whereas only a small fraction of granules traveled in directed fashion. High K(+) stimulation increased significantly the percentage of granules traveling in directed fashion. By dividing granules into the near-membrane group (within 1 microm from the plasma membrane) and cytosolic group, we have revealed significant differences between these two groups of granules in their mobility. The mobility of these two groups of granules is also differentially affected by disruption of F-actin, suggesting different mechanisms are involved in the motion of the two groups of granules. Our results demonstrate that combined deconvolution and single-particle tracking may find its application in three-dimensional tracking of long-term motion of granules and elucidating the underlying mechanisms.

  16. Epigenomics of Neural Cells: REST-Induced Down- and Upregulation of Gene Expression in a Two-Clone PC12 Cell Model

    PubMed Central

    Garcia-Manteiga, Jose M.; Bonfiglio, Silvia; Malosio, Maria Luisa; Lazarevic, Dejan; Stupka, Elia; Cittaro, Davide; Meldolesi, Jacopo

    2015-01-01

    Cell epigenomics depends on the marks released by transcription factors operating via the assembly of complexes that induce focal changes of DNA and histone structure. Among these factors is REST, a repressor that, via its strong decrease, governs both neuronal and neural cell differentiation and specificity. REST operation on thousands of possible genes can occur directly or via indirect mechanisms including repression of other factors. In previous studies of gene down- and upregulation, processes had been only partially investigated in neural cells. PC12 are well-known neural cells sharing properties with neurons. In the widely used PC12 populations, low-REST cells coexist with few, spontaneous high-REST PC12 cells. High- and low-REST PC12 clones were employed to investigate the role and the mechanisms of the repressor action. Among 15,500 expressed genes we identified 1,770 target and nontarget, REST-dependent genes. Functionally, these genes were found to operate in many pathways, from synaptic function to extracellular matrix. Mechanistically, downregulated genes were predominantly repressed directly by REST; upregulated genes were mostly governed indirectly. Among other factors, Polycomb complexes cooperated with REST for downregulation, and Smad3 and Myod1 participated in upregulation. In conclusion, we have highlighted that PC12 clones are a useful model to investigate REST, opening opportunities to development of epigenomic investigation. PMID:26413508

  17. Curcumin-Protected PC12 Cells Against Glutamate-Induced Oxidative Toxicity

    PubMed Central

    Chang, Chi-Huang; Chen, Hua-Xin; Yü, George

    2014-01-01

    Summary Glutamate is a major excitatory neurotransmitter present in the central nervous system. The glutamate/cystine antiporter system xc– connects the antioxidant defense with neurotransmission and behaviour. Overactivation of ionotropic glutamate receptors induces neuronal death, a pathway called excitotoxicity. Glutamate-induced oxidative stress is a major contributor to neurodegenerative diseases including cerebral ischemia, Alzheimer’s and Huntington’s disease. Curcuma has a wide spectrum of biological activities regarding neuroprotection and neurocognition. By reducing the oxidative damage, curcumin attenuates a spinal cord ischemia-reperfusion injury, seizures and hippocampal neuronal loss. The rat pheochromocytoma (PC12) cell line exhibits many characteristics useful for the study of the neuroprotection and neurocognition. This investigation was carried out to determine whether the neuroprotective effects of curcumin can be observed via the glutamate-PC12 cell model. Results indicate that glutamate (20 mM) upregulated glutathione peroxidase 1, glutathione disulphide, Ca2+ influx, nitric oxide production, cytochrome c release, Bax/Bcl-2 ratio, caspase-3 activity, lactate dehydrogenase release, reactive oxygen species, H2O2, and malondialdehyde; and downregulated glutathione, glutathione reductase, superoxide dismutase and catalase, resulting in enhanced cell apoptosis. Curcumin alleviates all these adverse effects. Conclusively, curcumin can effectively protect PC12 cells against the glutamate-induced oxidative toxicity. Its mode of action involves two pathways: the glutathione-dependent nitric oxide-reactive oxygen species pathway and the mitochondria-dependent nitric oxide-reactive oxygen species pathway. PMID:27904320

  18. The adaptor protein SH2B3 (Lnk) negatively regulates neurite outgrowth of PC12 cells and cortical neurons.

    PubMed

    Wang, Tien-Cheng; Chiu, Hsun; Chang, Yu-Jung; Hsu, Tai-Yu; Chiu, Ing-Ming; Chen, Linyi

    2011-01-01

    SH2B adaptor protein family members (SH2B1-3) regulate various physiological responses through affecting signaling, gene expression, and cell adhesion. SH2B1 and SH2B2 were reported to enhance nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells, a well-established neuronal model system. In contrast, SH2B3 was reported to inhibit cell proliferation during the development of immune system. No study so far addresses the role of SH2B3 in the nervous system. In this study, we provide evidence suggesting that SH2B3 is expressed in the cortex of embryonic rat brain. Overexpression of SH2B3 not only inhibits NGF-induced differentiation of PC12 cells but also reduces neurite outgrowth of primary cortical neurons. SH2B3 does so by repressing NGF-induced activation of PLCγ, MEK-ERK1/2 and PI3K-AKT pathways and the expression of Egr-1. SH2B3 is capable of binding to phosphorylated NGF receptor, TrkA, as well as SH2B1β. Our data further demonstrate that overexpression of SH2B3 reduces the interaction between SH2B1β and TrkA. Consistent with this finding, overexpressing the SH2 domain of SH2B3 is sufficient to inhibit NGF-induced neurite outgrowth. Together, our data demonstrate that SH2B3, unlike the other two family members, inhibits neuronal differentiation of PC12 cells and primary cortical neurons. Its inhibitory mechanism is likely through the competition of TrkA binding with the positive-acting SH2B1 and SH2B2.

  19. Ca2+ dynamics in the secretory vesicles of neurosecretory PC12 and INS1 cells.

    PubMed

    SantoDomingo, Jaime; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Moreno, Alfredo; Alvarez, Javier

    2010-11-01

    We have investigated the dynamics of the free [Ca(2+)] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca(2+)-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca(2+)] ([Ca(2+)](SG)] was around 20-40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca(2+) pumps with thapsigargin largely blocked Ca(2+) uptake by the granules in Ca(2+)-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca(2+) pump inhibitor benzohydroquinone induced a rapid release of Ca(2+) from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca(2+) pumps is essential to maintain the steady-state [Ca(2+)](SG). Both inositol 1,4,5-trisphosphate (InsP(3)) and caffeine produced a rapid Ca(2+) release from the granules, suggesting the presence of InsP(3) and ryanodine receptors in the granules. The response to high-K(+) depolarization was different in both cell types, a decrease in [Ca(2+)](SG) in PC12 cells and an increase in [Ca(2+)](SG) in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca(2+)](SG) in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca(2+) through SERCA-type Ca(2+) pumps and can release it through InsP(3) and ryanodine receptors, supporting the hypothesis that secretory granule Ca(2+) may be released during cell stimulation and contribute to secretion.

  20. Cobalt chloride induces neurite outgrowth in rat pheochromocytoma PC-12 cells through regulation of endothelin-2/vasoactive intestinal contractor.

    PubMed

    Kotake-Nara, Eiichi; Takizawa, Satoshi; Quan, Jiexia; Wang, Hongyu; Saida, Kaname

    2005-08-15

    We investigated whether endothelin-2/vasoactive intestinal contractor (ET-2/VIC) gene expression, upregulated by hypoxia in cancer cells, was associated with differentiation in neuronal cells. RT-PCR analysis, morphological observations, and immunostaining revealed that CoCl2, a hypoxic mimetic agent, at 200 microM increased expression of the ET-2/VIC gene, decreased expression of the ET-1 gene, and induced neurite outgrowth in PC-12 rat pheochromocytoma cells. These effects induced by 200 microM CoCl2 were completely inhibited by the antioxidant N-acetyl cysteine at 20 mM. In addition, CoCl2 increased the level of intracellular reactive oxygen species (ROS) at an early stage. Furthermore, interleukin (IL)-6 gene expression was upregulated upon the differentiation induced by CoCl2. These results suggest that expression of ET-2/VIC and ET-1 mediated by ROS may be associated with neuronal differentiation through the regulation of IL-6. When the cells were treated with 500 microM CoCl2 for 24 hr, however, ET-2/VIC gene expression disappeared, IL-6 gene expression was downregulated, and necrosis was subsequently induced in the PC-12 cells.

  1. Effects of selenocystine on lead-exposed Chinese hamster ovary (CHO) and PC-12 cells

    SciTech Connect

    Aykin-Burns, Nukhet; Ercal, Nuran . E-mail: nercal@umr.edu

    2006-07-15

    Lead is a pervasive environmental toxin that affects multiple organ systems, including the nervous, renal, reproductive, and hematological systems. Even though it is probably the most studied toxic metal, some of the symptoms of lead toxicity still cannot be explained by known molecular mechanisms. Therefore, lead-induced oxidative stress has recently started to gain attention. This in vitro study confirms the existence of oxidative stress due to lead exposure. Administration of lead acetate (PbA) to cultures of Chinese hamster ovary cells (CHO) had a concentration-dependent inhibitory effect on colony formation and cell proliferation. This inhibition was eliminated by 5 {mu}M selenocystine (SeCys). In order to evaluate the nature of SeCys's effect, we measured glutathione (GSH), its oxidized form glutathione disulfide (GSSG), malondialdehyde (MDA), catalase, and GSH peroxidase (GPx) activities in lead-exposed CHO cells both in the presence and absence of SeCys. Increases in MDA, catalase, and GPx activities were observed in cultures that received only PbA, but supplementation with SeCys returned these measures to pretreatment levels. The ratio of GSH to GSSG increased in lead-exposed cells incubated in SeCys-enhanced media but declined in cultures treated with PbA only. In order to determine whether SeCys also reverses lead-induced neurotoxicity, a neuronal cell line, PC-12 cells, was used. Lead's inhibition on neurite formation was significantly eliminated by SeCys in PC-12 cells. Our results suggest that SeCys can confer protection against lead-induced toxicity in CHO cells and neurotoxicity in PC-12 cells.

  2. Evaluation of antioxidant and cytoprotective activities of Artemisia ciniformis extracts on PC12 cells

    PubMed Central

    Mojarrab, Mahdi; Nasseri, Sajjad; Hosseinzadeh, Leila; Farahani, Farah

    2016-01-01

    Objective(s): In the current study antioxidant capacities of five different extracts of Artemisia ciniformis aerial parts were evaluated by cell-free methods. Then seven fractions of the potent extract were selected and their antioxidant capacity was assayed by cell free and cell based methods. Materials and Methods: Antioxidant ability was measured using the: 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test, β-carotene bleaching (BCB) method and ferrous ion chelating (FIC) assay. Total phenolic contents (TPC) of all the samples also were determined. The cytoprotective effect of fractions was evaluated by measuring the viability of cells after exposure to doxorubicin (DOX). The mechanism of action was studied by investigating caspase-3, mitochondrial membrane potential (MMP), the level of super-oxide dismutase (SOD) and intracellular reactive oxygen species (ROS). Results: Hydroethanolic extract exhibited a notably higher antioxidant activity and phenolic content. Among the fractions (A to G) of hydroethanolic extract, the highest antioxidant capacity was observed in the Fraction E. Moreover, 24 hr pretreatment of PC12 cells with fractions B, C and D decreased DOX-induced cytotoxicity. In addition, pre-treatment of cells with fraction B resulted in significant decrease in generation of the reactive oxygen species (ROS) and increase in the activity of SOD. We were able to demonstrate remarkable reduction in the activity of caspase-3 and increase in MMP in PC12 cells following pretreatment with fraction B. Conclusion: Our observations indicated that the fraction B of A. ciniformis hydroetanolic extract possessed protective effect on oxidative stress and apoptosis induced by DOX in PC12 cells. PMID:27279988

  3. Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells

    PubMed Central

    1989-01-01

    High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B- 50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B- 50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth- associated role for B-50, performed at the plasma membrane at the site of protrusion. PMID:2537833

  4. Partial Protection of PC12 Cells from Cellular Stress by Low-Dose Sodium Nitroprusside Pre-treatment.

    PubMed

    Varga, Judit; Bátor, Judit; Nádasdi, Gergő; Árvai, Zita; Schipp, Renáta; Szeberényi, József

    2016-10-01

    The PC12 rat pheochromocytoma cell line is an in vitro model system widely used for the investigation of intracellular signaling events contributing to neuronal differentiation and cell death. We found earlier that the nitric oxide donor compound sodium nitroprusside (SNP) induced apoptosis of PC12 cells if it was applied in high concentration (400 µM). Yoshioka et al. (J Pharmacol Sci 101:126-134, 2006) reported that cell death evoked by cytotoxic concentrations of SNP could be prevented by a 100 µM SNP pre-treatment in a murine macrophage cell line. The apoptosis caused by toxic-dose SNP treatment (400 µM) could be partially overcome in PC12 cells as well by the low-dose SNP pre-treatment. The partial inhibition of apoptosis was accompanied by reduced phosphorylation of certain proteins (such as stress-activated protein kinases, the p53, and the eIF2α proteins), decreased caspase activation, and less intense internucleosomal DNA fragmentation. The 100 µM SNP pre-treatment reduced the pro-apoptotic potential of certain other stress stimuli (serum withdrawal, cisplatin and tunicamycin treatments) as well, although the underlying biochemical changes were not entirely uniform. On the contrary, the 100 µM SNP pre-treatment was unable to prevent cell death caused by the protein synthesis inhibitor anisomycin. Further clarification of the above-mentioned processes may be important in understanding the mechanisms by which mild nitrosative stress protects cells against certain forms of cellular stress conditions.

  5. ADP ribosylation factor 1 is required for synaptic vesicle budding in PC12 cells.

    PubMed

    Faúndez, V; Horng, J T; Kelly, R B

    1997-08-11

    Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein-T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041-1049). A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

  6. Toxicity induced by cumene hydroperoxide in PC12 cells: protective role of thiol donors.

    PubMed

    Vimard, F; Saucet, M; Nicole, O; Feuilloley, M; Duval, D

    2011-01-01

    Oxidative shock and production of reactive oxygen species are known to play a major role in situations leading to neuron degeneration, but the precise mechanisms responsible for cell degeneration remain uncertain. In the present article, we have studied in PC 12 cells the effect of cumene hydroxyperoxide on both cell metabolism and morphology. We observed that relatively low concentrations of the drug (100 μM) led to a significant decrease in the cellular content of ATP and reduced glutathione as well as to a decreased mitochondrial potential. These metabolic alterations were followed by an important increase in intracellular free calcium and membrane disruption and death. In parallel, we observed profound changes in cell morphology with a shortening of cell extensions, the formation of ruffles and blebs at the cell surface, and a progressive detachment of the cells from the surface of the culture flasks. We also showed that addition of thiol donors such as N-acetylcysteine or β-mercaptoethanol, which were able to enhance cell glutathione content, almost completely protected PC 12 cells from the toxic action of cumene hydroperoxide whereas pretreatment by buthionine sulfoximine, a selective inhibitor of GSH synthesis, enhanced its action.

  7. Dopamine-melanin induces apoptosis in PC12 cells; possible implications for the etiology of Parkinson's disease.

    PubMed

    Offen, D; Ziv, I; Barzilai, A; Gorodin, S; Glater, E; Hochman, A; Melamed, E

    1997-08-01

    The function of neuromelanin (NM), the oxidized dopamine (DA) polymer, within the DA-producing cells in the human and primate substantia nigra (SN), is still an enigma. Some studies show that the vulnerability of nigral neurons in Parkinson's disease is correlated to their toxic NM content, while others suggest that it contributes to cellular protection. We showed recently that DA, the endogenous nigral neurotransmitter, triggers apoptosis, an active program of cellular self-destruction, in neuronal cultures. In the present study, we exposed cells to synthetic dopamine-melanin (DA-M) and analysed the cellular and genetic changes. We found that exposure of PC12 cells to DA-M (0.5 mg/ml for 24 h) caused 50% cell death, as indicated by trypan blue exclusion assay and 3H-thymidine incorporation. Gel electrophoresis DNA analysis of PC12 cells treated with DA-M showed the typical apoptotic DNA ladder, indicating inter-nucleosomal DNA degradation. The DNA fragmentation also was visualized histochemically in situ by DNA end-labeling staining (the TUNEL method). The FeCl2 (0.05 mM) significantly increased DA-M toxicity, while desferrioxamine, an iron chelator, totally abolished the additive toxicity of iron. The contribution of oxidative stress in this model of DA-M-induced cell death was examined using various antioxidants. In contrast to DA, inhibition of DA-M toxicity antioxidants by reduced glutathione (GSH), N-acetyl cysteine, catalase and Zn/Cu superoxide dismutase (SOD) was very limited. In conclusion, we found that DA-M may induce typical apoptotic death in PC12 cells. Our findings support a possible role of NM in the vulnerability of the dopaminergic neural degeneration in Parkinson's disease. The differential protective effect by antioxidants against toxicity of DA and DA-M may have implications for future neuroprotective therapeutic approaches for this common neurological disorder.

  8. Ascorbic acid protects against colistin sulfate-induced neurotoxicity in PC12 cells.

    PubMed

    Liu, Yang; Dai, Chongshan; Gao, Ruixia; Li, Jichang

    2013-10-01

    This study aimed to examine the protective effect of ascorbic acid against colistin-induced neurotoxicity mediated by oxidative stress, a potential mechanism. An in vitro neurotoxicity model was established with PC12 cells exposed to 125 µg/mL colistin sulfate for 24 h. PC12 cells were treated with colistin (125 µg/mL) in the absence and presence of ascorbic acid (0.1, 1.0 and 10 µM/mL) for 24 h. Both 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay were carried out to evaluate cell viability. The levels of intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione (GSH) levels were assessed. Moreover, we tested the level of DNA fragmentation, the release of cytochrome-c and the expressions of caspase-9 and -3 mRNA. The results showed that 1 and 10 µM/mL ascorbic acid significantly increased the cell viability and the levels of SOD and GSH (both p<0.05), while 0.1, 1 and 10 µM/mL ascorbic acid significantly decreased the generation of ROS, the release of cytochrome-c, formation of DNA fragmentation and the expressions of caspase-9 and -3 mRNA in colistin-treated PC12 cells, compared with the colistin model group. These results suggest that ascorbic acid could reduce colistin sulfate-induced neurotoxicity through the resistance of oxidative stress and the prevention of apoptosis mediated via mitochondria pathway. They also highlight the potential of coadministering ascorbic acid to widen the therapeutic dose of colistin.

  9. Reversible phosphorylation of tau to form A68 in heat-shocked neuronal PC12 cells.

    PubMed

    Wallace, W; Johnson, G; Sugar, J; Merril, C R; Refolo, L M

    1993-07-01

    A68, the primary protein constituent of Alzheimer's disease-associated neurofibrillary tangles, is an abnormally phosphorylated form of the microtubule-associated protein tau. We find that A68 is formed in neuronal PC12 cells when the cells are subjected to a heat shock (45 degrees C for 30 min). A68 was identified by immunoprecipitation with two different anti-tau antibodies (tau-2 and Alz50). Upon separation by SDS-polyacrylamide gel electrophoresis, the tau immunoprecipitates from heat-shocked cells exhibited an additional polypeptide of reduced electrophoretic mobility (approximately 68 kDa) when compared to control cells. A68 was formed with heat shock in the presence of cycloheximide, suggesting that its production occurred by post-translational modification of existing polypeptides. The tau/A68 polypeptides were identified as phosphoproteins by incorporation of 32P into the immunoprecipitates. The phosphorylation of tau to form A68 was reversed with recovery of the intact cells from the heat shock. Finally, immunoprecipitation of lysates from heat-shocked cells with antibodies to heat shock protein (hsp) 72/73 resulted in co-precipitation of tau with hsp 72, which indicates a stable complex formation between these two proteins. On the other hand, A68 remained unassociated with hsp during the heat shock. These results suggest that tau is reversibly phosphorylated to form A68 in neuronal PC12 cells under conditions of stress.

  10. Association of nerve growth factor receptors with the triton X-100 cytoskeleton of PC12 cells

    SciTech Connect

    Vale, R.D.; Ignatius, M.J.; Shooter, E.M.

    1985-10-01

    Triton X-100 solubilizes membranes of PC12 cells and leaves behind a nucleus and an array of cytoskeletal filaments. Nerve growth factor (NGF) receptors are associated with this Triton X-100-insoluble residue. Two classes of NGF receptors are found on PC12 cells which display rapid and slow dissociating kinetics. Although rapidly dissociating binding is predominant (greater than 75%) in intact cells, the majority of binding to the Triton X-100 cytoskeleton is slowly dissociating (greater than 75%). Rapidly dissociating NGF binding on intact cells can be converted to a slowly dissociating form by the plant lectin wheat germ agglutinin (WGA). This lectin also increases the number of receptors which associate with the Triton X-100 cytoskeleton by more than 10-fold. SVI-NGF bound to receptors can be visualized by light microscopy autoradiography in Triton X-100-insoluble residues of cell bodies, as well as growth cones and neurites. The WGA-induced association with the cytoskeleton, however, is not specific for the NGF receptor. Concentrations of WGA which change the Triton X-100 solubility of membrane glycoproteins are similar to those required to alter the kinetic state of the NGF receptor. Both events may be related to the crossbridging of cell surface proteins induced by this multivalent lectin.

  11. Effect of NGF on the subcellular localization of group IIA secretory phospholipase A(2) (GIIA) in PC12 cells: role in neuritogenesis.

    PubMed

    Ferrini, M; Nardicchi, V; Mannucci, R; Arcuri, C; Nicoletti, I; Donato, R; Goracci, G

    2010-12-01

    Phospholipases A(2) (PLA(2)s) are involved in neuritogenesis but the identity of the isoforms(s) contributing to this process is still not defined. Several reports have focused on secretory PLA(2)s (sPLA(2)) as the administration of exogenous sPLA(2)s to PC12 neuronal cells stimulates neurite outgrowth. The present study demonstrates that the endogenous group IIA sPLA(2) (GIIA), constitutively expressed in mammalian neural cells, changes its subcellular localization when PC12 cells are induced to differentiate by NGF treatment. Indeed, confocal analysis showed a time-dependent accumulation of GIIA in growth cones and neurite tips. Under identical conditions the subcellular distribution of another isoform (GV) was unaffected by NGF. Contrary to GX, another sPLA(2) isoform expressed by PC12 cells, the contribution of GIIA to neuritogenesis does not require its release in the extracellular medium.

  12. Pertussis toxin-insensitive effects of mastoparan, a wasp venom peptide, in PC12 cells.

    PubMed

    Murayama, T; Oda, H; Nomura, Y

    1996-12-01

    Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, modifies the secretion of neurotransmitters and hormones from a variety of cell types. Mastoparan interacts with heterotrimeric guanine nucleotide-binding proteins (G proteins) such as Gi and G(o), which are ADP-ribosylated by pertussis toxin (PTX) and thereby uncoupled from receptors. Previously, some of the effects of mastoparan including secretion were reported to be modified selectively by PTX but not by cholera toxin (CTX). In the present study, we examined the influence of bacterial toxins on the effects of mastoparan in PC12 cells. Mastoparan stimulated [3H]noradrenaline (NA) release from prelabeled PC12 cells in the absence of CaCl2, although high K+ or ATP-stimulated the release in a Ca(2+)-dependent manner. Pretreatment with CTX, not PTX, for 24 h inhibited mastoparan-stimulated [3H]NA release. Mastoparan inhibited forskolin-stimulated cyclic AMP accumulation in a dose-dependent manner, although mastoparan had no effect by itself. Pretreatment with PTX completely abolished the inhibitory effect of carbachol via Gi on cyclic AMP accumulation and partially reduced the effect of mastoparan. However, the inhibitory effect of 20 microM mastoparan was not modified by pretreatment with PTX. Thus, we investigated the effect of mastoparan on CTX-catalyzed [32P]ADP-ribosylation of proteins in PC12 cells. A subunit of CTX (CTX-A) catalyzed [32P]ADP-ribosylation of many proteins in the cytosolic fraction of PC12 cells. One of these was a 20 kDa protein, named ADP-ribosylating factor (ARF). The addition of mastoparan to assay mixtures inhibited ADP-ribosylation of many proteins including ARF and CTX-A in the presence of the cytosolic fraction. In the absence of the cytosolic fraction, however, mastoparan slightly enhanced ADP-ribosylation of bovine serum albumin and auto-ADP-ribosylation by CTX-A. Mastoparan did not inhibit ADP-ribosylation of the alpha subunit of Gs in the

  13. Phenolic compounds from Pueraria lobata protect PC12 cells against Aβ-induced toxicity.

    PubMed

    Choi, Yun-hyeok; Hong, Seong Su; Shin, Yu Su; Hwang, Bang Yeon; Park, So-Young; Lee, Dongho

    2010-10-01

    Bioassay-guided fractionation of the EtOAc-soluble extract of Pueraria lobata based on the inhibition of Aβ-induced toxicity in PC12 cells resulted in the isolation of four known active compounds, genistein (8), biochanin A (9), sissotrin (10), and puerol B (11). Of these, genistein (8) and biochanin A (9) exhibited potent neuroprotective effects with ED(50) values of 33.7 and 27.8 μM, respectively. In addition, a new coumestan, 2-(α,α-dimethylallyl)coumestrol (1) was isolated and characterized, but proved to be inactive, as were additional seven known compounds. The structure of new compound 1 was determined using spectroscopic techniques.

  14. Multi-walled carbon nanotubes suppress potassium channel activities in PC12 cells

    NASA Astrophysics Data System (ADS)

    Xu, Haifei; Bai, Juan; Meng, Jie; Hao, Wei; Xu, Haiyan; Cao, Ji-Min

    2009-07-01

    The advancement in nanotechnology has produced technological and conceptual breakthroughs but the effects nanomaterials have on organisms at the cellular level are poorly understood. Here we report that carboxyl-terminated multi-walled carbon nanotubes (MWCNTs) act as antagonists of three types of potassium channels as assessed by whole-cell patch clamp electrophysiology on undifferentiated pheochromocytoma (PC12) cells. Our results showed that carboxyl-terminated MWCNTs suppress the current densities of Ito, IK and IK1 in a time-dependent and irreversible manner. The suppressions were most distinct 24 h after incubation with MWCNTs. However, MWCNTs did not significantly change the expression levels of reactive oxygen species (ROS) or intracellular free calcium and also did not alter the mitochondrial membrane potential (ΔΨm) in PC12 cells. These results suggest that oxidative stress was not involved in the MWCNTs suppression of Ito, IK and IK1 current densities. Nonetheless, the suppression of potassium currents by MWCNTs will impact on electrical signaling of excitable cells such as neurons and muscles.

  15. Epidermal growth factor receptors on PC12 cells: alteration of binding properties by lectins

    SciTech Connect

    Vale, R.D.; Shooter, E.M.

    1983-01-01

    The PC12 cell line displays cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). It has been previously shown that the lectin wheat germ agglutinin (WGA) alters the properties of NGF receptors on these cells. We now report that preincubations with either WGA or concanavalin A (Con A) decrease the binding of /sup 125/I-EGF to PC12 cells by greater than 50%. The inhibition of binding occurred at 37 degrees C and 4 degrees C and could be blocked or reversed by the addition of sugars which bind specifically to WGA or Con A. Scatchard analysis revealed that these lectins decreased binding primarily by lowering the affinity of the receptor and to a lesser extent by decreasing receptor number. Succinylation of Con A (sCon A) produced a derivative that was less effective than the native lectin in decreasing EGF binding; however, addition of an antibody against Con A restored the ability of sCon A to decrease binding. Similar to results obtained with /sup 125/I-NGF binding, WGA but not Con A was found to increase, by severalfold, the proportion of /sup 125/I-EGF binding that is resistant to solubilization by Triton X-100 detergent. A potential association of the EGF receptor with cytoskeletal elements is discussed which could account for such results.

  16. Identification of coffee components that stimulate dopamine release from pheochromocytoma cells (PC-12).

    PubMed

    Walker, J; Rohm, B; Lang, R; Pariza, M W; Hofmann, T; Somoza, V

    2012-02-01

    Coffee and caffeine are known to affect the limbic system, but data on the influence of coffee and coffee constituents on neurotransmitter release is limited. We investigated dopamine release and Ca(2+)-mobilization in pheochromocytoma cells (PC-12 cells) after stimulation with two lyophilized coffee beverages prepared from either Coffea arabica (AR) or Coffea canephora var. robusta (RB) beans and constituents thereof. Both coffee lyophilizates showed effects in dilutions between 1:100 and 1:10,000. To identify the active coffee compound, coffee constituents were tested in beverage and plasma representative concentrations. Caffeine, trigonelline, N-methylpyridinium, chlorogenic acid, catechol, pyrogallol and 5-hydroxytryptamides increased calcium signaling and dopamine release, although with different efficacies. While N-methylpyridinium stimulated the Ca(2+)-mobilization most potently (EC(200): 0.14±0.29μM), treatment of the cells with pyrogallol (EC(200): 48±14nM) or 5-hydroxytryptamides (EC(200): 10±3nM) lead to the most pronounced effect on dopamine release. In contrast, no effect was seen for the reconstituted biomimetic mixture. We therefore conclude that each of the coffee constituents tested stimulated the dopamine release in PC-12 cells. Since no effect was found for their biomimetic mixture, we hypothesize other coffee constituents being responsible for the dopamine release demonstrated for AR and RB coffee brews.

  17. 5'-Aza-dC sensitizes paraquat toxic effects on PC12 cell.

    PubMed

    Kong, Min; Ba, Maowen; Liang, Hui; Ma, Lili; Yu, Quntao; Yu, Tianxia; Wang, Ying

    2012-08-22

    Parkinson's disease (PD) is one of the common neurodegenerative diseases that result in the progressive damage of dopaminergic neurons. Environmental exposure, including paraquat, is considered risky for PD. Epigenetics refer to the study of heritable changes in gene expression that occur without a change in DNA sequence. Epigenetic abnormalities (e.g. DNA methylation) have also been found to be causative factors in aging disease, such as PD. How these risk factors cooperate to induce progressive neurodegeneration in PD remains largely unknown. In this study, the PC12 cells was pretreated with methyltransferase inhibitor 5'-aza-2-deoxycytidi(5'-aza-dC) for 24 h, then exposed to paraquat for 12h. The biochemical mechanisms were investigated. The results showed that cell activity remarkably decreased and apoptotic cells increased after paraquat plus 5'-aza-dC treatment. Moreover, compared with paraquat treatment alone, after being exposed to paraquat plus 5'-aza-dC, the level of reactive oxygen species (ROS) increased significantly. The expression of bcl-2 decreased, expressions of bax increased, the rate of bcl-2/bax decreased, and thus expressions of cytochrome C increased. Our findings suggest that 5'-aza-dC modulating DNA methylation could sensitize paraquat toxic effects on PC12 cell by oxidative stress increment and mitochondrial deficit. Demonstration of the interaction of DNA methylation and paraquat provides additional new insights into the pathogenic mechanisms of PD.

  18. The herbal preparation Padma® 28 protects against neurotoxicity in PC12 cells.

    PubMed

    Ginsburg, Isaac; Rozenstein-Tsalkovich, Lea; Koren, Erez; Rosenmann, Hanna

    2011-05-01

    Padma® 28 is a multicompound herbal preparation based on the camphor formulas from traditional Tibetan medicine (TTM). It contains a variety of different secondary plant substances, which include terpenes and polyphenols such as flavonoids and tannins. As a rich source of antioxidant polyphenols, this herbal Padma 28 preparation seems to be a promising candidate for the treatment of degenerative diseases such as Alzheimer's disease (AD), a condition involving oxidative stress. Moreover, polyphenols have also been shown to mitigate AD neuropathology. The study investigated the protective effect of Padma 28 and of certain polyphenols on the neurotoxicity of PC12 cells induced by the neurotoxins: amyloid-beta (Aβ), glutamate, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 3-nitropropionate (3-NP), known to be involved in AD, Parkinson's disease (PD), amyotrophic-lateral-sclerosis (ALS) and Huntington's disease (HD), respectively. The decrease in cell viability induced by each of the toxins was significantly attenuated by Padma 28 treatment. Also, a decrease in the oxidative capacity of PC12 cells treated with Padma 28 was noted, indicating that the decrease in cell viability induced by the toxins might have been the result of an oxidative stress which could be attenuated by Padma 28 acting as a potent antioxidant. Padma 28, which is available in Europe and USA, seems to be a promising candidate for the treatment of CNS diseases.

  19. Protective Effects of Costunolide against Hydrogen Peroxide-Induced Injury in PC12 Cells.

    PubMed

    Cheong, Chong-Un; Yeh, Ching-Sheng; Hsieh, Yi-Wen; Lee, Ying-Ray; Lin, Mei-Ying; Chen, Chung-Yi; Lee, Chien-Hsing

    2016-07-09

    Oxidative stress-mediated cellular injury has been considered as a major cause of neurodegenerative diseases including Alzheimer's and Parkinson's diseases. The scavenging of reactive oxygen species (ROS) mediated by antioxidants may be a potential strategy for retarding the diseases' progression. Costunolide (CS) is a well-known sesquiterpene lactone, used as a popular herbal remedy, which possesses anti-inflammatory and antioxidant activity. This study aimed to investigate the protective role of CS against the cytotoxicity induced by hydrogen peroxide (H₂O₂) and to elucidate potential protective mechanisms in PC12 cells. The results showed that the treatment of PC12 cells with CS prior to H₂O₂ exposure effectively increased the cell viability. Furthermore, it decreased the intracellular ROS, stabilized the mitochondria membrane potential (MMP), and reduced apoptosis-related protein such as caspase 3. In addition, CS treatment attenuated the cell injury by H₂O₂ through the inhibition of phosphorylation of p38 and the extracellular signal-regulated kinase (ERK). These results demonstrated that CS is promising as a potential therapeutic candidate for neurodegenerative diseases resulting from oxidative damage and further research on this topic should be encouraged.

  20. Phosphatidylserine metabolism modification precedes manganese-induced apoptosis and phosphatidylserine exposure in PC12 cells.

    PubMed

    Ferrara, G; Gambelunghe, A; Mozzi, R; Marchetti, M C; Migliorati, G; Muzi, G; Buratta, S

    2013-12-01

    Long-term exposure to high manganese (Mn) levels can lead to Parkinson-like neurological disorders. Molecular mechanisms underlying Mn cytotoxicity have been not defined. It is known that Mn induces apoptosis in PC12 cells and that this involves the activation of some signal transduction pathways. Although the role of phospholipids in apoptosis and signal transduction is well-known, the membrane phospholipid component in Mn-related damage has not yet been investigated. Phosphatidylserine (PS) facilitates protein translocation from cytosol to plasma membrane and PS exposure on the cell surface allows macrophage recognition of apoptotic cells. This study investigates the effects of MnCl2 on PS metabolism in PC12 cells, relating them to those on cell apoptosis. Apoptosis induction decreased PS radioactivity of PC12 cells incubated with radioactive serine. MnCl2 reduced PS radioactivity even under conditions that did not affect cell viability or PS exposure, suggesting that the effects on PS metabolism may represent an early event in cell apoptosis. Thus the latter conditions that also induced a greater PS decarboxylation were utilized for further investigating on the effects on PS synthesis, by measuring the activity and expression of PS-synthesizing enzymes, in cell lysates and in total cellular membranes (TM). Compared with corresponding controls, enzyme activity of MnCl2-treated cells was lower in cell lysates and greater in TM. Evaluating the expression of two isoforms of PS-synthesizing enzyme (PSS), PSSII was increased both in cell lysate and TM, while PSSI was unchanged. MnCl2 addition to control cell lysate reduced enzyme activity. These results suggest Mn plays a dual role on PS synthesis. Once inside the cell, Mn inhibits the enzyme/s, thus accounting for reduced PS synthesis in lysates and intact cells. On the other hand, it increases PSSII expression in cell membranes. The possibility that this occurs to counteract the direct effects of Mn ions on enzyme

  1. Negletein as a neuroprotectant enhances the action of nerve growth factor and induces neurite outgrowth in PC12 cells.

    PubMed

    Phan, Chia-Wei; Sabaratnam, Vikineswary; Bovicelli, Paolo; Righi, Giuliana; Saso, Luciano

    2016-11-12

    Negletein has been shown to have therapeutic potential for inflammation-associated diseases, but its effect on neurite outgrowth is still unknown. The present study showed that negletein alone did not trigger PC12 cells to differentiate and extend neurites. When compared with the cells in the untreated control, a significant (P < 0.05) induction and a higher neurite outgrowth activity was observed when the cells were cotreated with negletein (10 µM) and a low dose of nerve growth factor (NGF; 5 ng/mL). The neurite outgrowth process was blocked by the tyrosine kinase receptor (Trk) inhibitor, K252a, suggesting that the neuritogenic effect was NGF-dependent. Negletein (10 µM) together with NGF (5 ng/mL) enhanced the phosphorylation of extracellular signal-regulated kinases (ERKs), protein kinase B (Akt), and cAMP response element-binding protein (CREB). The growth associated protein-43 (GAP-43) and the NGF level were also upregulated by negletein (10 µM) and a low dose of NGF (5 ng/mL). Negletein at nanomolar concentration also was found to be sufficient to mediate the survival of serum-deprived PC12 cells up to 72 h. Taken together, negletein might be useful as an efficient bioactive compound to protect neurons from cell death and promote neuritogenesis. © 2016 BioFactors, 42(6):591-599, 2016.

  2. A Simple HPLC-UV Method for the Determination of Glutathione in PC-12 Cells

    PubMed Central

    Appala, Raju N.; Appala, Raju V. V. S. S.

    2016-01-01

    A highly sensitive and simple HPLC-UV method was developed and validated for the assay of glutathione (GSH) in PC-12 cells. Glutathione is a major intracellular antioxidant having multiple biological effects, best known for its cytoprotective effects against cell damage from reactive oxygen species and toxic reactive metabolites and regulating the cellular redox homeostasis. Due to its own sulfhydryl (SH) group, GSH readily reacts with Ellman's reagent to form a stable dimer which allows for quantitative estimation of GSH in biological systems by UV detection. The separation was achieved using a C8 column with a mobile phase consisting of phosphate buffer adjusted to pH 2.5 (mobile phase A) and acetonitrile (mobile phase B), running in a segmented gradient manner at a flow rate of 0.8 mL/min, and UV detection was performed at 280 nm. The developed HPLC-UV method was validated with respect to precision, accuracy, robustness, and linearity within a range of 1–20 μg/mL. Limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 μg/mL, respectively. Furthermore, the method shows the applicability for monitoring the oxidative stress in PC-12 cells. PMID:27127683

  3. Cerebrosides from Sea Cucumber Protect Against Oxidative Stress in SAMP8 Mice and PC12 Cells.

    PubMed

    Che, Hongxia; Du, Lei; Cong, Peixu; Tao, Suyuan; Ding, Ning; Wu, Fengjuan; Xue, Changhu; Xu, Jie; Wang, Yuming

    2017-04-01

    Alzheimer's disease (AD) is a neurodegenerative disorder. Emerging evidence implicates β-amyloid (Aβ) plays a critical role in the progression of AD. In this study, we investigated the protective effect of cerebrosides obtained from sea cucumber against senescence-accelerated mouse prone 8 (SAMP8) mice in vivo. We also studied the effect of cerebrosides on Aβ-induced cytotoxicity on the rat pheochromocytoma cell (PC12) and the underlying molecular mechanisms. Cerebrosides ameliorated learning and memory deficits and the Aβ accumulation in demented mice, decreased the content of malondialdehyde (MDA), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG), 8-hydroxy-2'-deoxyguanosine (8-oxo-G), and nitric oxide (NO), and enhanced the superoxide dismutase (SOD) activity significantly. The neuroprotective effect of sea cucumber cerebrosides (SCC) was also verified in vitro: the cerebrosides increased the survival rate of PC12 cells, recovered the cellular morphology, downregulated the protein levels of Caspase-9, cleaved Caspase-3, total Caspase-3, and Bax, and upregulated the protein level of Bcl-2, revealing that cerebrosides could inhibit Aβ-induced cell apoptosis. The results showed the protective effect of SCC was regulated by the mitochondria-dependent apoptotic pathway. Our results provide a new approach to developing the marine organisms as functional foods for neuroprotection.

  4. Evaluation of silicon nitride as a substrate for culture of PC12 cells: an interfacial model for functional studies in neurons.

    PubMed

    Medina Benavente, Johan Jaime; Mogami, Hideo; Sakurai, Takashi; Sawada, Kazuaki

    2014-01-01

    Silicon nitride is a biocompatible material that is currently used as an interfacial surface between cells and large-scale integration devices incorporating ion-sensitive field-effect transistor technology. Here, we investigated whether a poly-L-lysine coated silicon nitride surface is suitable for the culture of PC12 cells, which are widely used as a model for neural differentiation, and we characterized their interaction based on cell behavior when seeded on the tested material. The coated surface was first examined in terms of wettability and topography using contact angle measurements and atomic force microscopy and then, conditioned silicon nitride surface was used as the substrate for the study of PC12 cell culture properties. We found that coating silicon nitride with poly-L-lysine increased surface hydrophilicity and that exposing this coated surface to an extracellular aqueous environment gradually decreased its roughness. When PC12 cells were cultured on a coated silicon nitride surface, adhesion and spreading were facilitated, and the cells showed enhanced morphological differentiation compared to those cultured on a plastic culture dish. A bromodeoxyuridine assay demonstrated that, on the coated silicon nitride surface, higher proportions of cells left the cell cycle, remained in a quiescent state and had longer survival times. Therefore, our study of the interaction of the silicon nitride surface with PC12 cells provides important information for the production of devices that need to have optimal cell culture-supporting properties in order to be used in the study of neuronal functions.

  5. ETAS, an enzyme-treated asparagus extract, attenuates amyloid beta-induced cellular disorder in PC12 cells.

    PubMed

    Ogasawara, Junetsu; Ito, Tomohiro; Wakame, Koji; Kitadate, Kentaro; Sakurai, Takuya; Sato, Shogo; Ishibashi, Yoshinaga; Izawa, Tetsuya; Takahashi, Kazuto; Ishida, Hitoshi; Takabatake, Ichiro; Kizaki, Takako; Ohno, Hideki

    2014-04-01

    One of the pathological characterizations of Alzheimer's disease (AD) is the deposition of amyloid beta peptide (Abeta) in cerebral cortical cells. The deposition of Abeta in neuronal cells leads to an increase in the production of free radicals that are typified by reactive oxygen species (ROS), thereby inducing cell death. A growing body of evidence now suggests that several plant-derived food ingredients are capable of scavenging ROS in mammalian cells. The purpose of the present study was to investigate whether enzyme-treated asparagus extract (ETAS), which is rich in antioxidants, is one of these ingredients. The pre-incubation of differentiated PC 12 cells with ETAS significantly recovered Abeta-induced reduction of cell viability, which was accompanied by reduced levels of ROS. These results suggest that ETAS may be one of the functional food ingredients with anti-oxidative capacity to help prevent AD.

  6. Protective effect of Hibiscus sabdariffa against serum/glucose deprivation-induced PC12 cells injury

    PubMed Central

    Bakhtiari, Elham; Hosseini, Azar; Mousavi, Seyed Hadi

    2015-01-01

    Objectives: Findings natural products with antioxidant and antiapoptotic properties has been one of the interesting challenges in the search for the treatment of neurodegenerative diseases including ischemic stroke. Serum/glucose deprivation (SGD) has been used as a model for the understanding of the molecular mechanisms of neuronal damage during ischemia in vitro and for the expansion of neuroprotective drugs against ischemia-induced brain injury. Recent studies showed that Hibiscus sabdariffa exert pharmacological actions such as potent antioxidant. Therefore, in this study we investigated the protective effect of extract of H. sabdariffa against SGD-induced PC12 cells injury. Materials and Methods: Cells were pretreated with different concentrations of H. sabdariffa extract (HSE) for 2 hr, and then exposed to SGD condition for 6, 12 and 18 hr. Results: SGD caused a major reduction in cell viability after 6, 12, and 18 hr as compared with control cells (p< 0.001). Pretreatment with HSE (30-500 𝜇g/mL) significantly increased cell viability following SGD insult for 6, 12 and 18 hr. A significant increase in cell apoptosis was seen in cells under SGD condition after 12hr as compared with control cells (p< 0.001). Pretreatment with HSE significantly decreased cell apoptosis subsequent SGD conditionafter12hr at concentration of 60, 125 and 250. Conclusion: These data showed that HSE had a protective property under SGD condition in PC12 cells, suggesting that H. sabdariffa has the potential to be used as a new therapeutic approach for neurodegenerative disorders. PMID:26101756

  7. Vincamine Alleviates Amyloid-β 25–35 Peptides-induced Cytotoxicity in PC12 Cells

    PubMed Central

    Han, Jianfeng; Qu, Qiumin; Qiao, Jin; Zhang, Jie

    2017-01-01

    Objective: Vincamine is a plant alkaloid used clinically as a peripheral vasodilator that increases cerebral blood flow and oxygen and glucose utilization by neural tissue to combat the effect of aging. The main purpose of the present study is to investigate the influence of vincamine on amyloid-β 25–35 (Aβ25–35) induced cytotoxicity, to gain a better understanding of the neuroprotective effects of this clinically used anti-Alzheimer's disease drug. Materials and Methods: Oxidative stress was assessed by measuring malondialdehyde, glutathione, and superoxide dismutase (SOD) levels. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis detection was performed using an Annexin-V-FITC Apoptosis Detection Kit. The production of reactive oxygen species (ROS) was determined using an ROS Assay Kit. Western blot detection was carried out to detect the protein expression. Results: Our studies showed that pretreatment with vincamine could reduce Aβ25–35 induced oxidative stress. Vincamine markedly inhibited cell apoptosis dose-dependently. More importantly, vincamine increased the phosphatidylinositol-3 kinase (PI3K)/Akt and Bcl-2 family protein ratios on preincubation with cells for 2 h. Conclusion: Above observation led us to assume that one possible mechanism of vincamine protects Aβ25-35-induced cell death could be through upregulation of SOD and activation of the PI3K/Akt pathway. SUMMARY Vincamine ameliorates amyloid-β 25–35 (Aβ25–35) peptides induced cytotoxicity in PC12 cellsVincamine reduces Aβ 25–35 peptides induced apoptosis of PC12 cellsVincamine activates the phosphatidylinositol-3 kinase/Akt pathwayVincamine up-regulates the superoxide dismutase. Abbreviation used: Aβ25-35: Amyloid-β 25-35; AD: Alzheimer's disease; BCA: Bicinchoninic acid; GSH: glutathione; PBS: Phosphate buffered solution; SDS: Sodium dodecylsulphate; SOD: Superoxide dismutase PMID:28216895

  8. Astragaloside IV Attenuates Glutamate-Induced Neurotoxicity in PC12 Cells through Raf-MEK-ERK Pathway

    PubMed Central

    Chen, Bingyang; Zhao, Jing; He, Weiwei; Yuan, Hu; Yuan, Xing; Gao, Na; Wu, Guozhen; Jin, Huizi; Shan, Lei; Zhang, Weidong

    2015-01-01

    Astragaloside IV (AGS-IV) is a main active ingredient of Astragalus membranaceus Bunge, a medicinal herb prescribed as an immunostimulant, hepatoprotective, antiperspirant, a diuretic or a tonic as documented in Chinese Materia Medica. In the present study, we employed a high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS to investigate the possible mechanism of action involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. Differential proteins were identified, among which 13 proteins survived the stringent filter criteria and were further included for functional discussion. Two proteins (vimentin and Gap43) were randomly selected, and their expression levels were further confirmed by western blots analysis. The results matched well with those of proteomics. Furthermore, network analysis of protein-protein interactions (PPI) and pathways enrichment with AGS-IV associated proteins were carried out to illustrate its underlying molecular mechanism. Proteins associated with signal transduction, immune system, signaling molecules and interaction, and energy metabolism play important roles in neuroprotective effect of AGS-IV and Raf-MEK-ERK pathway was involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. This study demonstrates that comparative proteomics based on shotgun approach is a valuable tool for molecular mechanism studies, since it allows the simultaneously evaluate the global proteins alterations. PMID:25961569

  9. Secretion of brain-derived neurotrophic factor from PC12 cells in response to oxidative stress requires autocrine dopamine signaling.

    PubMed

    Wang, Hong; Yuan, Guoxiang; Prabhakar, Nanduri R; Boswell, Mark; Katz, David M

    2006-02-01

    Expression of brain-derived neurotrophic factor (BDNF) is sensitive to changes in oxygen availability, suggesting that BDNF may be involved in adaptive responses to oxidative stress. However, it is unknown whether or not oxidative stress actually increases availability of BDNF by stimulating BDNF secretion. To approach this issue we examined BDNF release from PC12 cells, a well-established model of neurosecretion, in response to hypoxic stimuli. BDNF secretion from neuronally differentiated PC12 cells was strongly stimulated by exposure to intermittent hypoxia (IH). This response was inhibited by N-acetyl-l-cysteine, a potent scavenger of reactive oxygen species (ROS) and mimicked by exogenous ROS. IH-induced BDNF release requires activation of tetrodotoxin sensitive Na+ channels and Ca2+ influx through N- and L-type channels, as well as mobilization of internal Ca2+ stores. These results demonstrate that oxidative stress can stimulate BDNF release and that underlying mechanisms are similar to those previously described for activity-dependent BDNF secretion from neurons. Surprisingly, we also found that IH-induced secretion of BDNF was blocked by dopamine D2 receptor antagonists or by inhibition of dopamine synthesis with alpha-methyl-p-tyrosine. These data indicate that oxidative stress can stimulate BDNF release through an autocrine or paracrine loop that requires dopamine receptor activation.

  10. Altered expression and phosphorylation of amyloid precursor protein in heat shocked neuronal PC12 cells.

    PubMed

    Johnson, G; Refolo, L M; Merril, C R; Wallace, W

    1993-07-01

    The pathology of the Alzheimer's disease (AD) brain, including amyloid plaques, neurofibrillary tangles and neuronal degeneration, indicates that neurons affected by AD exist under conditions of stress. In fact, the brains of AD patients undergo many changes classically associated with the heat shock response, which is one form of a stress response. These changes include reduced protein synthesis, disrupted cytoskeleton, increased number of proteins associated with ubiquitin, and the induction of heat shock proteins. To investigate the response of neurons to stress, we examined neuronal PC12 cells incubated at either 37 degrees C (control cells) or 45 degrees C (heat-shocked cells). After a 30 min exposure at 45 degrees C, the heat-shocked cells exhibited several features characteristic of the classical heat shock response including a 45% reduction in total protein synthesis, the induction of heat shock protein 72, and an increased phosphorylation of the protein synthesis initiation factor eIF-2 alpha. We used this cellular model system to study the neuronal response to stress specifically focusing on protein synthesis elongation factor 2 (EF-2) and the Alzheimer's amyloid precursor protein (APP), the precursor form of beta-amyloid peptide. Hyperphosphorylation of EF-2 has been observed in the neocortex and hippocampus of AD brain. However, in our system, we find no hyperphosphorylation of EF-2 in response to heat shock. Heat-shocked neuronal PC12 cells exhibited two additional APP-like polypeptides not present in controls. We also found a significant decrease in the phosphorylation state of APP in response to heat shock.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Tl(I) and Tl(III) activate both mitochondrial and extrinsic pathways of apoptosis in rat pheochromocytoma (PC12) cells

    SciTech Connect

    Hanzel, Cecilia Eliana; Verstraeten, Sandra Viviana

    2009-04-01

    Thallium (Tl) is a highly toxic metal though yet its mechanisms are poorly understood. Previously, we demonstrated that rat pheochromocytoma (PC12) cells exposure to thallous (Tl(I)) or thallic (Tl(III)) cations leads to mitochondrial damage and reduced cell viability. In the present work we comparatively characterized the possible pathways involved in Tl(I)- and Tl(III)- (10-100 {mu}M) mediated decrease in PC12 cells viability. We observed that these cations do not cause cell necrosis but significantly increased the number of cells with apoptotic features. Both cations lead to Bax oligomerization and caused apoptosis inducing factor (AIF), endonuclease G (Endo G), and cytochrome c release from mitochondria, but they did not activate caspase dependent DNAse (CAD). Tl(I)- and Tl(III)-dependent caspases 9 and 3 activation followed similar kinetics, with maximal effects at 18 h of incubation. In addition, Tl(I) promoted phosphatidylserine (PS) exposure. Tl(III) induced 2- and 18-fold increase in Fas content and caspase 8 activity, respectively. Together, experimental results show that Tl(I) and Tl(III) induce PC12 cells apoptosis, although differential pathways are involved. While Tl(I)-mediated cell apoptosis was mainly associated with mitochondrial damage, Tl(III) showed a mixed effect triggering both the intrinsic and extrinsic pathways of apoptosis. These findings contribute to a better understanding of the mechanisms underlying Tl-induced loss of cell viability in PC12 cells.

  12. Calcium channel antagonist properties of the antineoplastic antiestrogen tamoxifen in the PC12 neurosecretory cell line

    SciTech Connect

    Greenberg, D.A.; Carpenter, C.L.; Messing, R.O.

    1987-01-01

    In view of existing evidence that Ca2+ may be important for tumor cell growth and metastasis, we investigated the effects of three antineoplastic drugs on K+-stimulated /sup 45/Ca2+ uptake through voltage-dependent Ca2+ channels of the PC12 neurosecretory cell line. The agents chosen for study (vinblastine, doxorubicin, and tamoxifen) were those previously shown to inhibit Ca2+/calmodulin- or Ca2+/phospholipid-activated protein kinases. Neither vinblastine nor doxorubicin altered /sup 45/Ca2+ uptake at concentrations that inhibit these Ca2+-dependent enzymes. However, tamoxifen reduced uptake (50% inhibitory dose, 8.6 +/- 0.9 (SE) microM) and competed for Ca2+ channel antagonist binding sites labeled by (/sup 3/H)-(+)PN200-110 (ki = 2.2 +/- 0.3 microM). Ca2+ channel antagonist properties may contribute to the effects of antineoplastic agents such as tamoxifen.

  13. Three-Dimensional Tracking of Single Granules in Living PC-12 Cells Employing TIRFM and WFFM.

    PubMed

    Xiong, Jun; Li, Dongdong; Zhu, Dan; Qu, Anlian

    2005-01-01

    A comparative study was carried out on evaluating the performance of total internal reflection fluorescence microscopy (TIRFM) and deconvolution wide-field fluorescence microscopy (WFFM) in tracking single secretory granules. Both techniques have been applied to follow the three-dimensional mobility of single secretory granules in living neuroendocrine PC-12 cells. Both techniques return the similar result that most acridine orange-labeled granules were found to travel in random and caged diffusion, and only a small fraction of granules traveled in directed diffusion. Furthermore, the size and 3-D diffusion coefficient of secretory granules, obtained by these two imaging techniques, yield the same value. Together, our results demonstrate the potential of the combination TIRFM and WFFM in tracking long-termed motion of granules throughout live whole cells.

  14. Screening of natural medicines that efficiently activate neurite outgrowth in PC12 cells in C2C12-cultured medium.

    PubMed

    Uezato, Tadayoshi; Sato, Eiji; Miura, Naoyuki

    2012-02-01

    We have studied the effects of natural medicines on neurite outgrowth in PC12D cells in a cultured medium of C2C12 cells. Derived from mouse myoblasts, the C2C12 cells secrete neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). The secretion of these neurotrophins from C2C12 cells stimulate neurite outgrowth in PC12D cells. We have screened a total of 120 samples and found five natural medicines: Trichosanthes Root, Asiasarum Root, Lycium Bark, Sinomenium Stem, and Dictamni radicis Cortex, that enhance the activity of C2C12-cultured medium to stimulate neurite outgrowth in PC12D cells. These natural medicines promoted not only neurite outgrowth but also stabilized the neurite formation in PC12D cells for several days. RT-PCR analysis showed that NGF was significantly increased with Trichosanthes and Lycium Bark. However, BDNF was slightly decreased with Lycium Bark, Sinomenium Stem, and Dictamni radicis Cortex. NT-3 was increased slightly by all of these natural medicines except Sinomenium Stem. All these five natural medicines significantly increased the number and length of neurites in PC12D cells in co-culture with C2C12 cells.

  15. Na+/Ca2+ exchanger inhibitors inhibit neurite outgrowth in PC12 cells.

    PubMed

    Oda, Toru; Kume, Toshiaki; Izumi, Yasuhiko; Ishihara, Kumatoshi; Sugmimoto, Hachiro; Akaike, Akinori

    2011-01-01

    To elucidate the role of Na(+)/Ca(2+) exchanger (NCX) in neurite outgrowth, we investigated the effects of NCX inhibitors on neurite outgrowth in PC12 cells. KB-R7943 and 3',4'-dichlorobenzamil, NCX inhibitors, inhibited the neurite outgrowth caused by nerve growth factor (NGF). NCX inhibitors inhibited the neurite outgrowth caused by dibutylyl cAMP, which rapidly reorganizes the cytoskeleton. KB-R7943 inhibited the neurite outgrowth caused by Y-27632, an inhibitor of Rho kinase (ROCK) that regulates actin. However, NCX inhibitors did not inhibit NGF-induced phosphorylation of extracellular signal-regulated kinase. These results suggest that NCX inhibitor affects downstream of the Rho-ROCK signal transduction pathways in neurite outgrowth.

  16. The cannabinoid beta-caryophyllene (BCP) induces neuritogenesis in PC12 cells by a cannabinoid-receptor-independent mechanism.

    PubMed

    Santos, Neife Aparecida Guinaim; Martins, Nádia Maria; Sisti, Flávia Malvestio; Fernandes, Laís Silva; Ferreira, Rafaela Scalco; de Freitas, Osvaldo; Santos, Antônio Cardozo

    2017-01-05

    Beta-caryophyllene (BCP) is a phytocannabinoid whose neuroprotective activity has been mainly associated with selective activation of cannabinoid-type-2 (CB2) receptors, inhibition of microglial activation and decrease of inflammation. Here, we addressed the potential of BCP to induce neuritogenesis in PC12 cells, a model system for primary neuronal cells that express trkA receptors, respond to NGF and do not express CB2 receptors. We demonstrated that BCP increases the survival and activates the NGF-specific receptor trkA in NGF-deprived PC12 cells, without increasing the expression of NGF itself. The neuritogenic effect of BCP in PC12 cells was abolished by k252a, an inhibitor of the NGF-specific receptor trkA. Accordingly, BCP did not induce neuritogenesis in SH-SY5Y neuroblastoma cells, a neuronal model that does not express trkA receptors and do not respond to NGF. Additionally, we demonstrated that BCP increases the expression of axonal-plasticity-associated proteins (GAP-43, synapsin and synaptophysin) in PC12 cells. It is known that these proteins are up-regulated by NGF in neurons and neuron-like cells, such as PC12 cells. Altogether, these findings suggest that BCP activates trka receptors and induces neuritogenesis by a mechanism independent of NGF or cannabinoid receptors. This is the first study to show such effects of BCP and their beneficial role in neurodegenerative processes should be further investigated.

  17. Secretory phospholipases A2 induce neurite outgrowth in PC12 cells.

    PubMed Central

    Nakashima, Satoru; Ikeno, Yutaka; Yokoyama, Tatsuya; Kuwana, Masakazu; Bolchi, Angelo; Ottonello, Simone; Kitamoto, Katsuhiko; Arioka, Manabu

    2003-01-01

    sPLA(2)s (secretory phospholipases A(2)) belong to a broad and structurally diverse family of enzymes that hydrolyse the sn -2 ester bond of glycerophospholipids. We previously showed that a secreted fungal 15 kDa protein, named p15, as well as its orthologue from Streptomyces coelicolor (named Scp15) induce neurite outgrowth in PC12 cells at nanomolar concentrations. We report here that both p15 and Scp15 are members of a newly identified group of fungal/bacterial sPLA(2)s. The phospholipid-hydrolysing activity of p15 is absolutely required for neurite outgrowth induction. Mutants with a reduced PLA(2) activity exhibited a comparable reduction in neurite-inducing activity, and the ability to induce neurites closely matched the capacity of various p15 forms to promote fatty acid release from live PC12 cells. A structurally divergent member of the sPLA(2) family, bee venom sPLA(2), also induced neurites in a phospholipase activity-dependent manner, and the same effect was elicited by mouse group V and X sPLA(2)s, but not by group IB and IIA sPLA(2)s. Lysophosphatidylcholine, but not other lysophospholipids, nor arachidonic acid, elicited neurite outgrowth in an L-type Ca(2+) channel activity-dependent manner. In addition, p15-induced neuritogenesis was unaffected by various inhibitors that block arachidonic acid conversion into bioactive eicosanoids. Altogether, these results delineate a novel, Ca(2+)- and lysophosphatidylcholine-dependent neurotrophin-like role of sPLA(2)s in the nervous system. PMID:12967323

  18. Developmental neurotoxicity of different classes of biocides in PC-12 cells in vitro.

    PubMed

    Christen, Verena; Rusconi, Manuel; Crettaz, Pierre; Fent, Karl

    2017-04-03

    The detection of developmental neurotoxicity (DNT) of chemicals has high relevance for protection of human health. However, DNT of many biocides is only little known. Furthermore, validated in vitro systems for assessment of DNT are not well established. Here we employed the rat phaeochromocytoma cell line PC-12 to evaluate DNT of 18 frequently used biocides of different classes, including neonicotinoids, pyrethroids, organophosphates, organochlorines, quaternary ammonium compounds, the biocidal active substance piperonylbutoxide, as well as the insect repellent DEET. We determined the outgrowth of neurites in PC-12 cells co-treated with nerve growth factor and different concentrations of biocides for 5days. Furthermore, we determined transcriptional alterations of selected genes that may be associated with DNT, such as camk2α and camk2β, gap-43, neurofilament-h, tubulin-α and tubulin-β. Strong and dose dependent inhibition of neurite outgrowth was induced by azamethiphos and chlorpyrifos, dieldrin and heptachlor, which was correlated with up-regulation of gap-43. No or only weak effects on neurite outgrowth and transcriptional alterations occurred for neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam, pyrethroids λ-cyhalothrin, cyfluthrin, deltamethrin, and permethrin, the biocidal disinfectants C12-C14-alkyl(ethylbenzyl)dimethylammonium (BAC), benzalkonium chloride and barquat (dimethyl benzyl ammonium chloride), and piperonylbutoxide and diethyltoluamide (DEET). Our study confirms potential developmental neurotoxicity of some biocides and provides first evidence that azamethiphos has the potential to act as a developmental neurotoxic compound. We also demonstrate that inhibition of neurite outgrowth and transcriptional alterations of gap-43 expression correlate, which suggests the employment of gap-43 expression as a biomarker for detection and initial evaluation of potential DNT of chemicals.

  19. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) induces initiation factor 2 alpha phosphorylation and translation inhibition in PC12 cells.

    PubMed

    Muñoz, F; Martín, M E; Salinas, M; Fando, J L

    2001-03-09

    We have investigated the effect of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on protein synthesis rate and initiation factor 2 (eIF2) phosphorylation in PC12 cells differentiated with nerve growth factor. FCCP treatment induced a very rapid 2-fold increase in intracellular Ca(2+) concentration that was accompanied by a strong protein synthesis rate inhibition (68%). The translation inhibition correlated with an increased phosphorylation of the alpha subunit of eIF2 (eIF2 alpha) (25% vs. 7%, for FCCP-treated and control cells, respectively) and a 1.7-fold increase in the double-stranded RNA-dependent protein kinase activity. No changes in the PKR endoplasmic reticulum-related kinase or eIF2 alpha phosphatase were found. Translational regulation may play a significant role in the process triggered by mitochondrial calcium mobilization.

  20. Non-cytotoxic Concentration of Cisplatin Decreases Neuroplasticity-Related Proteins and Neurite Outgrowth Without Affecting the Expression of NGF in PC12 Cells.

    PubMed

    Ferreira, Rafaela Scalco; Dos Santos, Neife Aparecida Guinaim; Martins, Nádia Maria; Fernandes, Laís Silva; Dos Santos, Antonio Cardozo

    2016-11-01

    Cisplatin is the most effective and neurotoxic platinum chemotherapeutic agent. It induces a peripheral neuropathy characterized by distal axonal degeneration that might progress to degeneration of cell bodies and apoptosis. Most symptoms occur nearby distal axonal branches and axonal degeneration might induce peripheral neuropathy regardless neuronal apoptosis. The toxic mechanism of cisplatin has been mainly associated with DNA damage, but cisplatin might also affect neurite outgrowth. Nevertheless, the neurotoxic mechanism of cisplatin remains unclear. We investigated the early effects of cisplatin on axonal plasticity by using non-cytotoxic concentrations of cisplatin and PC12 cells as a model of neurite outgrowth and differentiation. PC12 cells express NGF-receptors (trkA) and respond to NGF by forming neurites, branches and synaptic vesicles. For comparison, we used a neuronal model (SH-SY5Y cells) that does not express trkA nor responds to NGF. Cisplatin did not change NGF expression in PC12 cells and decreased neurite outgrowth in both models, suggesting a NGF/trkA independent mechanism. It also reduced axonal growth (GAP-43) and synaptic (synapsin I and synaptophysin) proteins in PC12 cells, without inducing mitochondrial damage or apoptosis. Therefore, cisplatin might affect axonal plasticity before DNA damage, NGF/trkA down-regulation, mitochondrial damage or neuronal apoptosis. This is the first study to show that neuroplasticity-related proteins might be early targets of the neurotoxic action of cisplatin and their role on cisplatin-induced peripheral neuropathy should be investigated in vivo.

  1. Osthole Attenuates Doxorubicin-Induced Apoptosis in PC12 Cells through Inhibition of Mitochondrial Dysfunction and ROS Production

    PubMed Central

    Shokoohinia, Yalda; Hosseinzadeh, Leila; Moieni-Arya, Maryam; Mostafaie, Ali; Mohammadi-Motlagh, Hamid-Reza

    2014-01-01

    Doxorubicin (DOX) is a potent, broad-spectrum chemotherapeutic drug used for treatment of several types of cancers. Despite its effectiveness, it has a wide range of toxic side effects, many of which most likely result from its inherent prooxidant activity. It has been reported that DOX has toxic effects on normal tissues, including brain tissue. In the current study, we investigated the protective effect of osthole isolated from Prangos ferulacea (L.) Lindl. on oxidative stress and apoptosis induced by DOX in PC12 as a neuronal model cell line. PC12 cells were pretreated with osthole 2 h after treatment with different concentrations of DOX. 24 h later, the cell viability, mitochondrial membrane potential (MMP), the activity of caspase-3, the expression ratio of Bax/Bcl-2, and the generation of intracellular ROS were detected. We found that pretreatment with osthole on PC12 cells significantly reduced the loss of cell viability, the activity of caspase-3, the increase in Bax/Bcl-2 ratio, and the generation of intracellular ROS induced by DOX. Moreover, pretreatment with osthole led to an increase in MMP in PC12 cells. In conclusion, our results indicated that pretreatment with nontoxic concentrations of osthole protected PC12 cells from DOX-mediated apoptosis by inhibition of ROS production. PMID:25013759

  2. Taurine inhibits 2,5-hexanedione-induced oxidative stress and mitochondria-dependent apoptosis in PC12 cells

    PubMed Central

    LI, Shuangyue; GUAN, Huai; QIAN, Zhiqiang; SUN, Yijie; GAO, Chenxue; LI, Guixin; YANG, Yi; PIAO, Fengyuan; HU, Shuhai

    2016-01-01

    2,5-hexanedione (HD) is the ultimate neurotoxic metabolite of hexane, causing the progression of nerve diseases in human. It was reported that HD induced apoptosis and oxidative stress. Taurine has been shown to be a potent antioxidant. In the present study, we investigated the protection of taurine against HD-induced apoptosis in PC12 cells and the underlying mechanism. Our results showed the decreased viability and increased apoptosis in HD-exposed PC12 cells. HD also induced the disturbance of Bax and Bcl-2 expression, the loss of MMP, the release of mitochondrial cytochrome c and caspase-3 activation in PC12 cells. Moreover, HD resulted in an increase in reactive oxygen species (ROS) level and a decline in the activities of superoxidedismutase and catalase in PC12 cells. However, taurine pretreatment ameliorated the increased apoptosis and the alterations in key regulators of mitochondria-dependent pathway in PC12 exposed to HD. The increased ROS level and the decreased activities of the antioxidant enzymes in HD group were attenuated by taurine. These results indicate that pretreatment of taurine may, at least partly, prevent HD-induced apoptosis via inhibiting mitochondria-dependent pathway. It is also suggested that the potential of taurine against HD-induced apoptosis may benefit from its anti-oxidative property. PMID:27840369

  3. p53 Mediates Colistin-Induced Autophagy and Apoptosis in PC-12 Cells

    PubMed Central

    Zhang, Ling; Xie, Daoyuan; Chen, Xueping; Hughes, Maria L. R.; Jiang, Guozheng; Lu, Ziyin; Xia, Chunli; Li, Li; Wang, Jinli; Xu, Wei; Sun, Yuan; Li, Rui; Wang, Rui; Qian, Feng

    2016-01-01

    The mechanism of colistin-induced neurotoxicity is still unknown. Our recent study (L. Zhang, Y. H. Zhao, W. J. Ding, G. Z. Jiang, Z. Y. Lu, L. Li, J. L. Wang, J. Li, and J. C. Li, Antimicrob Agents Chemother 59:2189–2197, 2015, http://dx.doi.org/10.1128/AAC.04092-14; H. Jiang, J. C. Li, T. Zhou, C. H. Wang, H. Zhang, and H. Wang, Int J Mol Med 33:1298–1304, 2014, http://dx.doi.org/10.3892/ijmm.2014.1684) indicates that colistin induces autophagy and apoptosis in rat adrenal medulla PC-12 cells, and there is interplay between both cellular events. As an important cellular stress sensor, phosphoprotein p53 can trigger cell cycle arrest and apoptosis and regulate autophagy. The aim of the present study was to investigate the involvement of the p53 pathway in colistin-induced neurotoxicity in PC-12 cells. Specifically, cells were treated with colistin (125 μg/ml) in the absence and presence of a p53 inhibitor, pifithrin-α (PFT-α; 20 nM), for 12 h and 24 h, and the typical hallmarks of autophagy and apoptosis were examined by fluorescence/immunofluorescence microscopy and electron microscopy, real-time PCR, and Western blotting. The results indicate that colistin had a stimulatory effect on the expression levels of the target genes and proteins involved in autophagy and apoptosis, including LC3-II/I, p53, DRAM (damage-regulated autophagy modulator), PUMA (p53 upregulated modulator of apoptosis), Bax, p-AMPK (activated form of AMP-activated protein kinase), and caspase-3. In contrast, colistin appeared to have an inhibitory effect on the expression of p-mTOR (activated form of mammalian target of rapamycin), which is another target protein in autophagy. Importantly, analysis of the levels of p53 in the cells treated with colistin revealed an increase in nuclear p53 at 12 h and cytoplasmic p53 at 24 h. Pretreatment of colistin-treated cells with PFT-α inhibited autophagy and promoted colistin-induced apoptosis. This is the first study to demonstrate that colistin

  4. Rotenone induces KATP channel opening in PC12 cells in association with the expression of tyrosine hydroxylase.

    PubMed

    Bai, Qunhua; He, Junlin; Qiu, Jingfu; Wang, Yang; Wang, Shibo; Xiu, Yun; Yu, Chao

    2012-10-01

    The activation of ATP-sensitive potassium (KATP) channels in PC12 cells play a pivotal role in protection against the neurotoxic effect of rotenone. However, it remains unclear why rotenone seems to preferentially affect activation of KATP channels and if this could affect its physiological activity. In this study, we sought to determine how the different energy states caused by various doses of rotenone affect the KATP opening state and whether the KATP opening state influences the expression of tyrosine hydroxylase (TH) which is related with DA synthesis. With patch clamp technology, results showed that treatment of PC12 cells with rotenone (0.2-1 µg/ml) for 15 min can cause KATP channel opening with significantly increased intracellular ROS production. Treatment with rotenone (2-16 ng/ml) for 24 h also caused the channels to open with gently increased ROS. In order to study if the rather long-term action on KATP channel opening states could affect the specified function of PC12 cells, the KATP channel opener pinacidil and the inhibitor glibenclamide were used to treat cells for 24 h, and the expression of TH was detected. Our results showed that treatment of PC12 cells with glibenclamide for 24 h can notably promote TH expression and can also enhance the expression of TH which were reduced by rotenone. These data indicate that the energy states in PC12 induced by various doses of rotenone could significantly influence the opening states of KATP channels. However long-term energy stress may raise the opening rate and opening sensitivity of this channel. In addition, our results demonstrate for the first time that activation of plasma membrane KATP channels induced by rotenone inhibits TH expression which influences DA synthesis in PC12 cells.

  5. Regulation of Fusion Pore Closure and Compound Exocytosis in Neuroendocrine PC12 Cells by SCAMP1

    PubMed Central

    Zhang, Jie; Castle, David

    2011-01-01

    During exocytosis, neuroendocrine cells can achieve partial release of stored secretory products from dense core vesicles (DCVs) by coupling endocytosis directly at fusion sites and without full discharge. The physiological role of partial secretion is of substantial interest. Much is known about SNARE-mediated initiation of exocytosis and dynamin-mediated completion of endocytosis, but little is known about coupling events. We have used real-time microscopy to examine the role of secretory carrier membrane protein SCAMP1 in exo-endocytic coupling in PC12 cells. While reduced SCAMP1 expression is known to impede dilation of newly opened fusion pores during onset of DCV exocytosis, we now show that SCAMP1 deficiency also inhibits closure of fusion pores after they have opened. Inhibition causes accumulation of fusion figures at the plasma membrane. Closure is recovered by restoring expression and accelerated slightly by overexpression. Interestingly, inhibited pore closure resulting from loss of SCAMP1 appears to increase secondary fusion of DCVs to already-fused DCVs (compound exocytosis). Unexpectedly, reinternalization of expanded DCV membranes following compound exocytosis appears to proceed normally in SCAMP1-deficient cells. SCAMP1’s apparent dual role in facilitating dilation and closure of fusion pores implicates its function in exo-endocytic coupling and in the regulation of partial secretion. Secondarily, SCAMP1 may serve to limit the extent of compound exocytosis. PMID:21272170

  6. The Association of VDAC with Cell Viability of PC12 Model of Huntington’s Disease

    PubMed Central

    Karachitos, Andonis; Grobys, Daria; Kulczyńska, Klaudia; Sobusiak, Adrian; Kmita, Hanna

    2016-01-01

    It is becoming increasingly apparent that mitochondria dysfunction plays an important role in the pathogenesis of Huntington’s disease (HD), but the underlying mechanism is still elusive. Thus, there is a still need for further studies concerning the upstream events in the mitochondria dysfunction that could contribute to cell death observed in HD. Taking into account the fundamental role of the voltage-dependent anion-selective channel (VDAC) in mitochondria functioning, it is reasonable to consider the channel as a crucial element in HD etiology. Therefore, we applied inducible PC12 cell model of HD to determine the relationship between the effect of expression of wild type and mutant huntingtin (Htt and mHtt, respectively) on cell survival and mitochondria functioning in intact cells under conditions of undergoing cell divisions. Because after 48 h of Htt and mHtt expression differences in mitochondria functioning co-occurred with differences in the cell viability, we decided to estimate the effect of Htt and mHtt expression lasted for 48 h on VDAC functioning. Therefore, we isolated VDAC from the cells and tested the preparations by black lipid membrane system. We observed that the expression of mHtt, but not Htt, resulted in changes of the open state conductance and voltage-dependence when compared to control cells cultured in the absence of the expression. Importantly, for all the VDAC preparations, we observed a dominant quantitative content of VDAC1, and the quantitative relationships between VDAC isoforms were not changed by Htt and mHtt expression. Thus, Htt and mHtt-mediated functional changes of VDAC, being predominantly VDAC1, which occur shortly after these protein appearances in cells, may result in differences concerning mitochondria functioning and viability of cells expressing Htt and mHtt. The assumption is important for better understanding of cytotoxicity as well as cytoprotection mechanisms of potential clinical application. PMID

  7. Protective effects of nimodipine and lithium against aluminum-induced cell death and oxidative stress in PC12 cells

    PubMed Central

    Saberzadeh, Jamileh; Omrani, Mehdi; Takhshid, Mohammad Ali

    2016-01-01

    Objective(s): The role of aluminum (Al) in the pathogenesis of neurodegenerative diseases has been implicated in several studies. However, the exact mechanisms of cytotoxic effects of Al have not been elucidated yet. The aim of this study was to investigate the effect of L-type calcium channel antagonist, nimodipine (NM), and lithium chloride (LiCl) on Al-induced toxicity in PC12 cells. Materials and Methods: PC12 cells were treated with Al-maltolate (Almal) in the presence and absence of different concentrations of NM (50-150 μm) and/or LiCl (0.5-1.0 mM) for 48 hr. Cell viability, apoptosis, and catalase (CAT) activity, a marker of oxidative stress, were then measured using MTT, flow cytometry and enzyme assay, respectively. Results: The results showed that Almal, dose dependently induced cell death, apoptosis and CAT activity in the PC12 cells. NM significantly increased cell viability and decreased apoptosis and CAT activity of Almal-treated cells in a dose dependent mode. LiCl reduced CAT activity and increased cell viability in Almal-treated cells, without significant effect on apoptosis (P=0.74). Conclusion: These findings suggest that NM and Li may have benefits in the prevention of Al-induced cytotoxicity through decreasing oxidative stress. PMID:27917283

  8. Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells.

    PubMed Central

    Lang, T; Wacker, I; Wunderlich, I; Rohrbach, A; Giese, G; Soldati, T; Almers, W

    2000-01-01

    In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 x 10(-4) microm(2)/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg(2+) and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion. PMID:10827968

  9. A dual function for Munc-18 in exocytosis of PC12 cells.

    PubMed

    Schütz, Dagmar; Zilly, Felipe; Lang, Thorsten; Jahn, Reinhard; Bruns, Dieter

    2005-05-01

    Munc-18 interacts with the SNARE protein syntaxin and is supposed to influence transmitter release by controlling the formation of exocytosis-relevant SNARE complexes. Here, we used combined biochemical and physiological analyses to study the role of the Munc-18/syntaxin interaction in large dense core vesicle (LDCV) exocytosis of neuroendocrine PC12 cells. We compared two Munc-18 mutants carrying mutations in the syntaxin-binding region and show that Munc-18's membrane association depends on direct binding to syntaxin. The data suggest that perturbation of syntaxin binding inhibits neurotransmitter release upstream of the individual fusion event implying an essential role of the Munc-18/syntaxin complex leading to exocytosis. Furthermore, we show that a Munc-18 mutant lacking any syntaxin binding has a stimulatory effect on secretion, and provide evidence that the Munc-18/Mint1 interaction may constitute a second pathway for Munc-18 to regulate exocytosis. We propose that Munc-18 represents a dynamic link between syntaxin-related and Mint1-related mechanisms, both involved in the control of LDCV exocytosis in neuroendocrine cells.

  10. Ethanol's effects on neurotransmitter release and intracellular free calcium in PC12 cells

    SciTech Connect

    Rabe, C.S.; Weight, F.F.

    1988-01-01

    The effect of ethanol on muscarine-stimulated release of (/sup 3/H)NE was studied using the rat pheochromocytoma cell line, PC12. At concentrations of 25 mM and above, ethanol produced a dose dependent inhibition of muscarine-stimulated release of (/sup 3/H)NE. The inhibition of muscarine-stimulated transmitter release occurred in the absence of any effect of ethanol on (/sup 3/H)NE uptake, metabolism or on muscarinic binding to the cells. However, ethanol produced an inhibition of muscarine-stimulated elevation of intracellular free Ca2+ which corresponded with the inhibition of transmitter release. At concentrations greater than 100 mM, ethanol produced both a stimulation of the release of (/sup 3/H)NE as well as an increase in intracellular free Ca2+. The increase in basal transmitter release and intracellular free Ca2+ occurred independent of the inhibition by ethanol of muscarine-stimulated elevation of intracellular free Ca2+ or transmitter section. These results demonstrate the relationship of the effects of ethanol on cellular free Ca2+ and neurotransmitter release.

  11. Effects of ethanol on neurotransmitter release and intracellular free calcium in PC12 cells

    SciTech Connect

    Rabe, C.S.; Weight, F.F.

    1988-02-01

    The effect of ethanol on muscarine-stimulated release of l-(/sup 3/H)norepinephrine ((/sup 3/H)NE) was studied using the rat pheochromocytoma cell line, PC12. At concentrations of 25 mM and above, ethanol produced a dose-dependent inhibition of muscarine-stimulated release of (/sup 3/H)NE. The inhibition of muscarine-stimulated transmitter release occurred in the absence of any detectable effect of ethanol on (/sup 3/H)NE uptake or on muscarinic binding to the cells. However, ethanol produced an inhibition of muscarine-stimulated elevation of intracellular free Ca++ which corresponded with the inhibition of transmitter release. At concentrations greater than 100 mM, ethanol produced an increase in the basal release of (/sup 3/H)NE. Intracellular free Ca++ also was increased by ethanol concentrations greater than 100 mM. The elevation of basal transmitter release and intracellular free Ca++ by concentrations of ethanol greater than 100 mM occurred independently of the inhibition by ethanol of muscarine-stimulated elevation of intracellular free Ca++ and transmitter secretion. These results suggest that the effects of ethanol on neurotransmitter release are associated with the effects of ethanol on intracellular free Ca++.

  12. Three-dimensional co-culture of C2C12/PC12 cells improves skeletal muscle tissue formation and function.

    PubMed

    Ostrovidov, Serge; Ahadian, Samad; Ramon-Azcon, Javier; Hosseini, Vahid; Fujie, Toshinori; Parthiban, S Prakash; Shiku, Hitoshi; Matsue, Tomokazu; Kaji, Hirokazu; Ramalingam, Murugan; Bae, Hojae; Khademhosseini, Ali

    2017-02-01

    Engineered muscle tissues demonstrate properties far from native muscle tissue. Therefore, fabrication of muscle tissues with enhanced functionalities is required to enable their use in various applications. To improve the formation of mature muscle tissues with higher functionalities, we co-cultured C2C12 myoblasts and PC12 neural cells. While alignment of the myoblasts was obtained by culturing the cells in micropatterned methacrylated gelatin (GelMA) hydrogels, we studied the effects of the neural cells (PC12) on the formation and maturation of muscle tissues. Myoblasts cultured in the presence of neural cells showed improved differentiation, with enhanced myotube formation. Myotube alignment, length and coverage area were increased. In addition, the mRNA expression of muscle differentiation markers (Myf-5, myogenin, Mefc2, MLP), muscle maturation markers (MHC-IId/x, MHC-IIa, MHC-IIb, MHC-pn, α-actinin, sarcomeric actinin) and the neuromuscular markers (AChE, AChR-ε) were also upregulated. All these observations were amplified after further muscle tissue maturation under electrical stimulation. Our data suggest a synergistic effect on the C2C12 differentiation induced by PC12 cells, which could be useful for creating improved muscle tissue. Copyright © 2014 John Wiley & Sons, Ltd.

  13. Protective effect of Nigella sativa extract and thymoquinone on serum/glucose deprivation-induced PC12 cells death.

    PubMed

    Mousavi, S H; Tayarani-Najaran, Z; Asghari, M; Sadeghnia, H R

    2010-05-01

    The serum/glucose deprivation (SGD)-induced cell death in cultured PC12 cells represents a useful in vitro model for the study of brain ischemia and neurodegenerative disorders. Nigella sativa L. (family Ranunculaceae) and its active component thymoquinone (TQ) has been known as a source of antioxidants. In the present study, the protective effects of N. sativa and TQ on cell viability and reactive oxygen species (ROS) production in cultured PC12 cells were investigated under SGD conditions. PC12 cells were cultured in DMEM medium containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 microg/ml streptomycin. Cells were seeded overnight and then deprived of serum/glucose for 6 and 18 h. Cells were pretreated with different concentrations of N. sativa extract (15.62-250 microg/ml) and TQ (1.17-150 microM) for 2 h. Cell viability was quantitated by MTT assay. Intracellular ROS production was measured by flow cytometry using 2',7'-dichlorofluorescin diacetate (DCF-DA) as a probe. SGD induced significant cells toxicity after 6, 18, or 24 h (P < 0.001). Pretreatment with N. sativa (15.62-250 microg/ml) and TQ (1.17-37.5 microM) reduced SGD-induced cytotoxicity in PC12 cells after 6 and 18 h. A significant increase in intracellular ROS production was seen following SGD (P < 0.001). N. sativa (250 microg/ml, P < 0.01) and TQ (2.34, 4.68, 9.37 microM, P < 0.01) pretreatment reversed the increased ROS production following ischemic insult. The experimental results suggest that N. sativa extract and TQ protects the PC12 cells against SGD-induced cytotoxicity via antioxidant mechanisms. Our findings might raise the possibility of potential therapeutic application of N. sativa extract and TQ for managing cerebral ischemic and neurodegenerative disorders.

  14. Azole fungicides disturb intracellular Ca2+ in an additive manner in dopaminergic PC12 cells.

    PubMed

    Heusinkveld, Harm J; Molendijk, Jeffrey; van den Berg, Martin; Westerink, Remco H S

    2013-08-01

    Humans are exposed to complex mixtures of pesticides and other compounds, mainly via food. Azole fungicides are broad spectrum antifungal compounds used in agriculture and in human and veterinary medicine. The mechanism of antifungal action relies on inhibition of CYP51, resulting in inhibition of fungal cell growth. Known adverse health effects of azole fungicides are mainly linked to CYP inhibition. Additionally, azole fungicide-induced neurotoxicity has been reported, though the underlying mechanism(s) are largely unknown. We therefore investigated the effects of a group of six azole fungicides (imazalil, flusilazole, fluconazole, tebuconazole, triadimefon, and cyproconazole) on cell viability using a combined alamar Blue/CFDA-AM assay and on oxidative stress using a H2-DCFDA fluorescent assay. As calcium plays a pivotal role in neuronal survival and functioning, effects of these six azole fungicides and binary and quaternary mixtures of azole fungicides on the intracellular calcium concentration ([Ca(2+)]i) were investigated using single-cell fluorescence microscopy in dopaminergic PC12 cells loaded with the calcium-sensitive fluorescent dye Fura-2. Only modest changes in cell viability and ROS production were observed. However, five out of six azole fungicides induced a nonspecific inhibition of voltage-gated calcium channels (VGCCs), though with varying potency. Experiments using binary IC20 and quaternary IC10 mixtures indicated that the inhibitory effects on VGCCs are additive. The combined findings demonstrate modulation of intracellular Ca(2+) via inhibition of VGCCs as a novel mode of action of azole fungicides. Furthermore, mixtures of azole fungicides display additivity, illustrating the need to take mixture effects into account in human risk assessment.

  15. Extracellular norepinephrine reduces neuronal uptake of norepinephrine by oxidative stress in PC12 cells.

    PubMed

    Mao, Weike; Qin, Fuzhong; Iwai, Chikao; Vulapalli, Raju; Keng, Peter C; Liang, Chang-seng

    2004-07-01

    Cardiac norepinephrine (NE) uptake activity is reduced in congestive heart failure. Our studies in intact animals suggest that this effect on the cardiac sympathetic nerve endings is caused by oxidative stress and/or NE toxic metabolites derived from NE. In this study, we investigated the direct effects of NE on neuronal NE uptake activity and NE transporter (NET), using undifferentiated PC12 cells. Cells were incubated with NE (1-500 microM) either alone or in combination of Cu(2+) sulfate (1 microM), which promotes free radical formation by Fenton reaction for 24 h. NE uptake activity was measured using [(3)H]NE. Cell viability was determined with the use of Trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay, and cellular oxidative stress by dichlorodihydrofluorescein fluorescence and the GSH/GSSG ratio. Cell viability was reduced by NE >100 microM. At lower doses, NE produced oxidative stress and a dose-dependent reduction of NE uptake activity without affecting cell viability significantly. Cu(2+), which has no direct effect on NE uptake activity, potentiated oxidative stress and reduction of NE uptake activity produced by NE. This decrease of NE uptake activity was associated with reductions of NE uptake binding sites and NET protein expression by using the radioligand assay and Western blot analysis, but no changes in NET gene expression. In addition, the free-radical scavenger mannitol, and antioxidant enzymes superoxide dismutase and catalase, reduced oxidative stress and attenuated the reductions of NE uptake activity and NET protein produced by NE/Cu. Thus our results support a functional role of oxidative stress in mediating the neuronal NE uptake reducing effect of NE and that this effect of NE on NET is a posttranscriptional event.

  16. Postconditioning mitigates cell death following oxygen and glucose deprivation in PC12 cells and forebrain reperfusion injury in rats.

    PubMed

    Lin, Han-Chen; Narasimhan, Purnima; Liu, Shin-Yun; Chan, Pak H; Lai, I-Rue

    2015-01-01

    Postconditioning mitigates ischemia-induced cellular damage via a modified reperfusion procedure. Mitochondrial permeability transition (MPT) is an important pathophysiological change in reperfusion injury. This study explores the role of MPT modulation underlying hypoxic postconditioning (HPoC) in PC12 cells and studies the neuroprotective effects of ischemic postconditioning (IPoC) on rats. Oxygen-glucose deprivation (OGD) was performed for 10 hr on PC12 cells. HPoC was induced by three cycles of 10-min reoxygenation/10-min rehypoxia after OGD. The MPT inhibitor N-methyl-4-isoleucine cyclosporine (NIM811) and the MPT inducer carboxyatractyloside (CATR) were administered to selective groups before OGD. Cellular death was evaluated by flow cytometry and Western blot analysis. JC-1 fluorescence signal was used to estimate the mitochondrial membrane potential (△Ψm ). Transient global cerebral ischemia (tGCI) was induced via the two-vessel occlusion and hypotension method in male Sprague Dawley rats. IPoC was induced by three cycles of 10-sec reperfusion/10-sec reocclusion after index ischemia. HPoC and NIM811 administration attenuated cell death, cytochrome c release, and caspase-3 activity and maintained △Ψm of PC12 cells after OGD. The addition of CATR negated the protection conferred by HPoC. IPoC reduced neuronal degeneration and cytochrome c release and cleaved caspase-9 expression of hippocampal CA1 neurons in rats after tGCI. HPoC protected PC12 cells against OGD by modulating the MPT. IPoC attenuated degeneration of hippocampal neurons after cerebral ischemia.

  17. Cobalt chloride induces PC12 cells apoptosis through reactive oxygen species and accompanied by AP-1 activation.

    PubMed

    Zou, W; Yan, M; Xu, W; Huo, H; Sun, L; Zheng, Z; Liu, X

    2001-06-15

    Reactive oxygen species (ROS) are supposed to play an important role in hypoxia- and ischemia/reperfusion-mediated neuronal injury with the characteristics of apoptosis. There are many reports showing that cobalt chloride (CoCl(2)) could mimic the hypoxic responses in some aspects including production of ROS in cultured cells. The cytotoxicity of CoCl(2) and its molecular mechanisms have yet to be elucidated. We report that CoCl(2) triggered neuronal PC12 cells apoptosis in a dose- and time-dependent manner. Apoptosis was demonstrated by morphological changes and DNA fragmentation, and was dependent on macromolecular synthesis. Apoptosis was also confirmed by the decrease of the expression of Bcl-X(L). To our knowledge, this is the first documentation of the apoptotic induction of CoCl(2) on PC12 cells. Furthermore, ROS production in PC12 cells was increased during CoCl(2) treatment. Antioxidants, which could inhibit ROS production, significantly blocked CoCl(2)-induced apoptosis, suggesting that apoptosis is mediated by ROS production. We also observed a significant increase of the DNA-binding activity of AP-1 in response to CoCl(2) and this increase was blocked by antioxidants, showing that CoCl(2)-induced apoptosis is accompanied by ROS-activated AP-1. CoCl(2)-treated PC12 cells may serve as an in vitro model for studies of molecular mechanisms in ROS-linked neuronal disorders.

  18. Arachidonic acid release and prostaglandin F(2alpha) formation induced by anandamide and capsaicin in PC12 cells.

    PubMed

    Someya, Akiyoshi; Horie, Syunji; Murayama, Toshihiko

    2002-08-23

    Anandamide, an endogenous agonist of cannabinoid receptors, activates various signal transduction pathways. Anandamide also activates vanilloid VR(1) receptor, which was a nonselective cation channel with high Ca(2+) permeability and had sensitivity to capsaicin, a pungent principle in hot pepper. The effects of anandamide and capsaicin on arachidonic acid metabolism in neuronal cells have not been well established. We examined the effects of anandamide and capsaicin on arachidonic acid release in rat pheochromocytoma PC12 cells. Both agents stimulated [3H]arachidonic acid release in a concentration-dependent manner from the prelabeled PC12 cells even in the absence of extracellular CaCl(2). The effect of anandamide was neither mimicked by an agonist nor inhibited by an antagonist for cannabinoid receptors. The effects of anandamide and capsaicin were inhibited by phospholipase A(2) inhibitors, but not by an antagonist for vanilloid VR(1) receptor. In PC12 cells preincubated with anandamide or capsaicin, [3H]arachidonic acid release was marked and both agents were no more effective. Co-addition of anandamide or capsaicin synergistically enhanced [3H]arachidonic acid release by mastoparan in the absence of CaCl(2). Anandamide stimulated prostaglandin F(2alpha) formation. These findings suggest that anandamide and capsaicin stimulated arachidonic acid metabolism in cannabinoid receptors- and vanilloid VR(1) receptor-independent manner in PC12 cells. The possible mechanisms are also discussed.

  19. Protective effects of veskamide, enferamide, becatamide, and oretamide on H2O2-induced apoptosis of PC-12 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Veskamide, enferamide, becatamide, and oretamide are phenolic amides whose analogues are found in plants. In this study, the four amides were prepared by chemical synthesis and their protective effects on H(2)O(2)-induced apoptosis in PC-12 cells were investigated. The syntheses were relatively si...

  20. Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells

    PubMed Central

    1985-01-01

    The outgrowth of neurites from rat PC12 cells stimulated by combined treatment of nerve growth factor (NGF) with cAMP is significantly more rapid and extensive than the outgrowth induced by either factor alone. We have compared the responses of PC12 cells under three different growth conditions, NGF alone, cAMP alone, and combined treatment, with respect to surface morphology, rapidity of neurite outgrowth, and stability of neurite microtubules, to understand the synergistic action of NGF and cAMP on PC12. Surface events at early times in these growth conditions varied, suggesting divergent pathways of action of NGF and cAMP. This suggestion is strongly supported by the finding that cells exposed to saturating levels of dibutyryl cAMP without substantial neurite outgrowth initiated neurites within 5 min of NGF. This response has been adopted as a convenient assay for NGF. Neurites that regenerated in the three growth conditions showed marked differences in stability to treatments that depolymerize microtubules. The results indicate that microtubules in cells treated with both NGF and cAMP are significantly more stable than in either growth factor alone. We suggest that a shift of the assembly equilibrium favoring tubulin assembly is a necessary prerequisite for the initiation of neurites by PC12. PMID:2982887

  1. MAGNETIC FIELD INFLUENCE ON NGF-STIMULATED NEURITE OUTGROWTH IN PC-12 CELLS: EFFECT OF PAINT FUMES

    EPA Science Inventory

    MAGNETIC FIELD INFLUENCE ON NGF-STIMULATED NEURITE OUTGROWTH IN PC-12 CELLS: EFFECT OF PAINT FUMES. C. F. Blackman1, D. E. House2*, S. G. Benane3*, A. Ubeda4, M.A. TrilIo4. 1 National Health and Environmental Effects Research Laboratory, EPA,
    Research Triangle Park, North Caro...

  2. MELATONIN-INDUCED SUPPRESSION OF PC12 CELL GROWTH IS MEDIATED BY ITS GI COUPLED TRANSMEMBRANE RECEPTORS. (R826248)

    EPA Science Inventory

    The effects of pertussis toxin, an uncoupler of Gi protein from adenylate cyclase, and luzindole, a competitive inhibitor of melatonin receptor binding, were examined for their ability to inhibit melatonin-induced suppression of PC12 cell growth. Both agents inhibited the mela...

  3. Effects of tobacco smoke on PC12 cell neurodifferentiation are distinct from those of nicotine or benzo[a]pyrene.

    PubMed

    Slotkin, Theodore A; Card, Jennifer; Stadler, Ashley; Levin, Edward D; Seidler, Frederic J

    2014-01-01

    Although nicotine accounts for a great deal of the neurodevelopmental damage associated with maternal smoking or second-hand exposure, tobacco smoke contains thousands of potentially neurotoxic compounds. We used PC12 cells, a standard in vitro model of neurodifferentiation, to compare tobacco smoke extract (TSE) to nicotine, matching TSE exposure (with its inherent nicotine content) to parallel concentrations of nicotine, or to benzo[a]pyrene, a tobacco combustion product. TSE promoted the transition from cell replication to differentiation, resulting in fewer, but larger cells with greater neurite extension. TSE also biased differentiation into the dopaminergic versus the cholinergic phenotype, evidenced by an increase in tyrosine hydroxylase activity but not choline acetyltransferase. Nicotine likewise promoted differentiation at the expense of cell numbers, but its effect on growth and neurite extension was smaller than that of TSE; furthermore, nicotine did not promote the dopaminergic phenotype. Benzo[a]pyrene had effects opposite to those of TSE, retarding neurodifferentiation, which resulted in higher cell numbers, smaller cells, reduced neurite information, and impaired emergence of both dopaminergic and cholinergic phenotypes. Our studies show that the complex mixture of compounds in tobacco smoke exerts direct effects on neural cell replication and differentiation that resemble those of nicotine in some ways but not others, and most importantly, that are greater in magnitude than can be accounted for from just the nicotine content of TSE. Thus, fetal tobacco smoke exposure, including lower levels associated with second-hand smoke, could be more injurious than would be anticipated from measured levels of nicotine or its metabolites.

  4. Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

    SciTech Connect

    Lee, Jeong Eun; Park, Jae Hyeon; Shin, In Chul; Koh, Hyun Chul

    2012-09-01

    Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity as well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.

  5. Neuroprotective activities of catalpol against CaMKII-dependent apoptosis induced by LPS in PC12 cells

    PubMed Central

    Chen, Wenna; Li, Ximing; Jia, Lian-Qun; Wang, Jun; Zhang, Lin; Hou, Diandong; Wang, Junyan; Ren, Lu

    2013-01-01

    Background and Purpose Neurodegenerative diseases present progressive neurological disorder induced by cell death or apoptosis. Catalpol, an iridoid glucoside isolated from the root of Rehmannia glutinosa Libosch, is present in a wide range of plant families. Although catalpol is an effective anti-apoptotic agent in LPS-induced neurodegeneration, the underlying mechanism has not been established. Here we have identified some of the mechanisms involved the prevention by catalpol of apoptosis induced by LPS in an experimental model of neurodegeneration in vitro. Experimental Approach Apoptosis was induced by adding LPS (80 ng·mL−1) to pheochromocytoma (PC12) cells, pretreated with catalpol for 12 h. We measured intracellular reactive oxygen species (ROS), apoptosis and intracellular calcium concentration ( [Ca2+]i) by flow cytometry or laser confocal scanning microscopy. We also analysed the protein expression of Bcl-2, Bax and Ca2+-calmodulin-dependent protein kinase II (CaMKII)-dependent apoptosis signal-regulating kinase-1 (ASK-1)/JNK/p38 signalling pathway in PC12 cells by Western blot. Key Results Catalpol stimulated expression of Bcl-2 and inhibited the expression of Bax. Catalpol also attenuated the increase in Ca2+ concentration induced by LPS in PC12 cells and down-regulated CaMK phosphorylation. The CaMKII-dependent ASK-1/JNK/p38 signalling cascade was blocked by catalpol. All these changes were accompanied by a decrease of apoptosis induced by LPS in PC12 cells. Conclusions and Implications The data presented here provide new mechanistic insights into the links between the CaMKII-dependent ASK-1/JNK/p38 signalling pathway and the protective effect of catalpol on apoptosis induced by LPS in PC12 cells. PMID:23550774

  6. Effect of chloroquine on biosynthesis, processing and secretion of proteins from PC12 cells

    SciTech Connect

    Sarmalkar, M.; Kuhn, L.J.; Sabban, E.L.

    1986-05-01

    Chloroquine is a lysomotropic agent that can raise intraorganelle pH, and has been proposed to divert secretion from a regulated to a constitutive pathway. The authors examined the effect of chloroquine on biosynthesis of dopamine ..beta..-hydroxylase (DBH) in PC12 pheochromocytoma cells. DBH is normally present as a 77,000-Mr and a 73,000-Mr subunit form in near equal amounts. The 77K membrane-bound form is a precursor of the 73K soluble form, which can be secreted with norepinephrine. Pretreatment for 1 hr with 50 ..mu..M -1 mM chloroquine and labelling in its presence for 4 hrs inhibited protein synthesis by approx. 50% with 200 ..mu..M and approx. 90% with 1 mM chloroquine. The overall profile of proteins synthesized was unaltered. However, in the presence of 200 ..mu..M chloroquine, the 73K form of DBH predominated. Thus, chloroquine enhanced the post-translational processing of the 77K to the 73K form. Endoglycosidase H digestion of the 73K form from chloroquine-treated or untreated cells yielded a 67.3 K product. Treatment with 200 ..mu..M and 1 mM chloroquine essentially prevented the release of (/sup 35/S)Met-labeled proteins which normally accompany the release of norepinephrine, and allowed the stimulated release of a new set of proteins (<68,000 daltons). The results are very similar to those obtained with monensin. Thus, elevation in intraorganelle pH appears to enhance processing of DBH and impede the secretory process.

  7. Distinct signalling particles containing ERK/MEK and B-Raf in PC12 cells.

    PubMed

    MacCormick, Matt; Moderscheim, Tanja; van der Salm, Louise W M; Moore, Anna; Pryor, Shona Clements; McCaffrey, Gretchen; Grimes, Mark L

    2005-04-01

    Although several multiprotein complexes containing MAPKs (mitogen-activated protein kinases) have been identified using overexpression of kinases and scaffold proteins, the components of the complexes and their physical properties at endogenous expression levels have not been defined. We characterized a large protein complex containing a nerve-growth-factor-activated ERK (extracellular-signal-regulated kinase) and MEK (MAPK/ERK kinase) in rat pheochromocytoma (PC12) cells. This protein complex fractionated into a high-speed pellet and was resistant to non-ionic detergent treatments that solubilized membranes. Disruption of protein-protein interactions by treatment with high salt was required to facilitate immunoprecipitation of active ERK1 and co-precipitation of MEK1. Microtubule fragments were also present in the detergent-resistant high-speed pellet, and some kinases were bound to them, especially ERK1b (an alternatively spliced isoform of ERK1), which showed a strong preference for binding microtubules. The large protein complex containing ERK1 and MEK1 was resolved by velocity sedimentation from fragments of microtubules; however, it did not contain other scaffolding components known to bind ERK and MEK. B-Raf was also present in a distinct detergent-resistant, microtubule-independent protein complex slightly larger than that containing ERK and MEK. We conclude that there are two independent nerve growth factor-regulated 'signalling particles' with an estimated size of 60-75 S, one containing ERK1 and MEK1 and the other containing B-Raf. These signalling particles may have a role in the temporal and spatial regulation of kinase activity inside cells.

  8. Distinct signalling particles containing ERK/MEK and B-Raf in PC12 cells

    PubMed Central

    MacCORMICK, Matt; Moderscheim, Tanja; van der Salm, Louise W. M.; Moore, Anna; Pryor, Shona Clements; McCAFFREY, Gretchen; Grimes, Mark L.

    2004-01-01

    Although several multiprotein complexes containing MAPKs (mitogen-activated protein kinases) have been identified using overexpression of kinases and scaffold proteins, the components of the complexes and their physical properties at endogenous expression levels have not been defined. We characterized a large protein complex containing a nerve-growth-factor-activated ERK (extracellular-signal-regulated kinase) and MEK (MAPK/ERK kinase) in rat pheochromocytoma (PC12) cells. This protein complex fractionated into a high-speed pellet and was resistant to non-ionic detergent treatments that solubilized membranes. Disruption of protein–protein interactions by treatment with high salt was required to facilitate immunoprecipitation of active ERK1 and co-precipitation of MEK1. Microtubule fragments were also present in the detergent-resistant high-speed pellet, and some kinases were bound to them, especially ERK1b (an alternatively spliced isoform of ERK1), which showed a strong preference for binding microtubules. The large protein complex containing ERK1 and MEK1 was resolved by velocity sedimentation from fragments of microtubules; however, it did not contain other scaffolding components known to bind ERK and MEK. B-Raf was also present in a distinct detergent-resistant, microtubule-independent protein complex slightly larger than that containing ERK and MEK. We conclude that there are two independent nerve growth factor-regulated ‘signalling particles’ with an estimated size of 60–75 S, one containing ERK1 and MEK1 and the other containing B-Raf. These signalling particles may have a role in the temporal and spatial regulation of kinase activity inside cells. PMID:15500439

  9. Photosensitizer-induced fluorescence of the rat adrenal gland and rat pheochromocytoma cells (PC 12) by meso-tetra(hydroxyphenyl)chlorin (mTHPC)

    NASA Astrophysics Data System (ADS)

    Colombo-Benkmann, Mario; Muhm, Markus; Gahlen, Johannes; Heym, Christine; Senninger, Norbert

    1997-12-01

    Rat adrenal glands exhibit an intense mTHPC-induced fluorescence. The objective of our study was the identification of adrenal cells exhibiting mTHPC-induced fluorescence under normal conditions and under stimulation of adrenal proliferation by reserpine. Furthermore mTHPC-uptake of rat pheochromocytoma (PC 12) cells was investigated. Four male Wistar rats received 0.5 mg mTHPC/kg iv 48 hours before perfusion. Furthermore four rats received reserpine (2 mg/kg im od), bromo-deoxy-uridine (BrdU; 50 mg/kg ip od) each for one week and mTHPC (0.5 mg/kg) 48 hours before perfusion. BrdU was detected immunohistochemically. PC 12-cells were incubated with 0.5 mg mTHPC/l culture medium for 24 or 48 hours. Cells and tissues were examined by fluorescence microscopy. The adrenal cortex exhibited an intense mTHPC-induced fluorescence. The adrenal medulla fluoresced faintly. Reserpine increased fluorescence of intramedullary cells, not coinciding with adrenal proliferation. Cortical fluorescence remained unchanged. PC 12-cells lying singly or in small groups and differentiating cells showed a more intense mTHPC- induced fluorescence than confluent cells. Differences of cortical and medullary uptake of mTHPC are independent of proliferation and may be explained by lipophilia of mTHPC, since adrenocytes have an uptake mechanism for cholesterol. The difference of mTHPC-uptake between PC 12-cells and chromaffin cells implicate the possibility of photodynamic applications for medullary neoplasia.

  10. A non-muscle myosin II motor links NR1 to retrograde trafficking and proteasomal degradation in PC12 cells.

    PubMed

    Vazhappilly, Rema; Wee, Karen Siaw-Ling; Sucher, Nikolaus J; Low, Chian-Ming

    2010-03-01

    Rat pheochromocytoma (PC12) cells have been shown to lack functional NMDA receptors; yet, these cells express NR1 subunits of the NMDA receptor. The reason for the lack of functional receptors has been attributed to the absence of significant levels of NR2 subunits to co-assemble with NR1. It is known that PC12 expresses very low levels of NR2C, with complete absence of other types of NR2 subunits. The purpose of the present study is to describe the molecular mechanism of trafficking and degradation of unassembled NR1 subunits in PC12 cells. The localization of NR1 subunits in PC12 cells were evaluated by immunofluorescence and co-immunoprecipitation, which showed that NR1 was present in the endoplasmic reticulum and cis-middle compartments of the Golgi apparatus. Upon treatment with a proteasome inhibitor, MG132, the ubiquitinylated species of NR1 subunit were detected, suggesting that NR1 is being targeted for endoplasmic reticulum-associated proteasomal degradation. Our previous studies suggest that NR1 subunits from the Golgi do not proceed to trans-Golgi, hence they will require re-routing to the endoplasmic reticulum for degradation. Further investigations on the factors involved in the trafficking of NR1 from Golgi to endoplasmic reticulum were performed using co-immunoprecipitation and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. These revealed the co-association of NR1 with non-muscle myosin heavy chain II isoforms A and B. We also demonstrate the functional significance of this interaction through the use of a myosin inhibitor, blebbistatin, to disrupt brefeldin A-induced Golgi-to-endoplasmic reticulum trafficking of NR1. In conclusion, our results suggest that non-muscle myosin II is involved in the retrograde trafficking of NR1 subunits from the cis/middle-Golgi to the endoplasmic reticulum for proteasomal degradation in PC12.

  11. Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA

    PubMed Central

    1993-01-01

    We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPA-treated cells were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation- independent mRNA stabilization mechanism. PMID:8436593

  12. Nerve growth factor induced changes in the Golgi apparatus of PC-12 rat pheochromocytoma cells as studied by ligand endocytosis, cytochemical and morphometric methods.

    PubMed

    Hickey, W F; Stieber, A; Hogue-Angeletti, R; Gonatas, J; GOnatas, N K

    1983-10-01

    Cells of the PC-12 rat pheochromocytoma cell line respond to nerve growth factor (NGF) by sprouting neurites and biochemically differentiating into sympathetic ganglion-like cells. NGF-stimulated ('differentiated') and unstimulated ('undifferentiated') cells were studied by cytochemical techniques for the localization of the enzymes acid phosphatase (ACPase) and thiamine pyrophosphatase (TPPase), and by a morphometric analysis of the distribution of endocytosed wheat-germ agglutinin labelled with horseradish peroxidase (WGA-HRP). Both cytochemical stains showed the enzymes to be distributed in lysosomes and certain cisternae of the Golgi apparatus in both NGF stimulated and unstimulated cells. ACPase was not confined to GERL (Golgi-endoplasmic reticulum-lysosome) as in certain other cells. The morphometric studies demonstrated that the reaction product of the internalized WGA-HRP occupied 4.7% of the cytoplasmic area in unstimulated cells and 4.5% in NGF-stimulated ones. Despite this similarity, the distribution of the WGA-HRP among the studied intracellular compartments in these two cell groups varied. In the NGF-stimulated cells 3.3% of the WGA-HRP reaction product was found in the innermost Golgi cisterna(e) while in unstimulated cells only 0.3% was seen in this compartment. Similarly, 4.3% of the WGA-HRP stain was found in small vesicles at the 'trans' aspect of the Golgi apparatus in stimulated cells, when only 0.3% of the stain occupied this compartment in 'undifferentiated' cells. The morphometric analysis also revealed that when the PC-12 cells were stimulated with NGF, the Golgi apparatus increased in area by approximately 70%. These findings are consistent with the hypothesis that NGF induced differentiation of PC-12 cells is coupled with enhanced endocytosis of WGA and probably of its 'receptor' to the innermost Golgi cisterna(e) and the closely associated vesicles.

  13. Knockdown of Dystrophin Dp71 Impairs PC12 Cells Cycle: Localization in the Spindle and Cytokinesis Structures Implies a Role for Dp71 in Cell Division

    PubMed Central

    Villarreal-Silva, Marcela; Centeno-Cruz, Federico; Suárez-Sánchez, Rocío; Garrido, Efraín; Cisneros, Bulmaro

    2011-01-01

    The function of dystrophin Dp71 in neuronal cells remains to be established. Previously, we revealed the involvement of this protein in both nerve growth factor (NGF)-induced neuronal differentiation and cell adhesion by isolation and characterization of PC12 neuronal cells with depleted levels of Dp71. In this work, a novel phenotype of Dp71-knockdown cells was characterized, which is their delayed growth rate. Cell cycle analyses revealed an altered behavior of Dp71-depleted cells, which consists of a delay in G0/G1 transition and an increase in apoptosis during nocodazole-induced mitotic arrest. Dp71 associates with lamin B1 and β-dystroglycan, proteins involved in aspects of the cell division cycle; therefore, we compared the distribution of Dp71 with that of lamin B1 and β-dystroglycan in PC12 cells at mitosis and cytokinesis by means of immunofluorescence and confocal microscopy analysis. All of these three proteins exhibited a similar immunostaining pattern, localized at mitotic spindle, cleavage furrow, and midbody. It is noteworthy that a drastic decreased staining in mitotic spindle, cleavage furrow, and midbody was observed for both lamin B1 and β-dystroglycan in Dp71-depleted cells. Furthermore, we demonstrated the interaction of Dp71 with lamin B1 in PC12 cells by immunoprecipitation and pull-down assays, and importantly, we revealed that knockdown of Dp71 expression caused a marked reduction in lamin B1 levels and altered localization of the nuclear envelope protein emerin. Our data indicate that Dp71 is a component of the mitotic spindle and cytokinesis multi-protein apparatuses that might modulate the cell division cycle by affecting lamin B1 and β-dystroglycan levels. PMID:21886794

  14. Electron probe X-ray microanalysis of cisplatin-induced cell death in rat pheochromocytoma PC12 cells.

    PubMed

    Ramos, Juan M; Arrebola, Francisco; Fernández-Cervilla, Francisco J; Crespo, Vicente; Fernández-Segura, Eduardo

    2011-03-01

    Several lines of evidence suggest that cisplatin-induced cell death is not always the result of apoptosis. A distinctive feature between apoptosis and necrosis is the alteration in cell volume regulation and ion homeostasis. Here we analyzed the changes in intracellular element content during cell death induced by exposure to therapeutic concentrations of cisplatin in the PC12 cell line. To quantitate Na, Cl and K content, electron probe X-ray microanalysis (EPXMA) was performed in whole freeze-dried cells. We also traced the alterations in morphological features with fluorescence and transmission electron microscopy. EPXMA demonstrated progressive derangement of the absolute intracellular Na, Cl and K contents. Cisplatin-treated cells showed two microanalytical patterns: 1) cells with alterations in elemental content typical of apoptosis, i.e., an increase in intracellular Na and a decrease in intracellular Cl and K, and 2) cells characterized by an increase in Na content and a decrease in K content, with no changes in Cl content. This intracellular profile for Na, Cl, and K was not typical of necrosis or apoptosis. Morphological analysis revealed two cellular phenotypes: 1) cells characterized by a phenotype typical of apoptosis, and 2) cells characterized by a hybrid phenotype combining variable features of apoptosis and necrosis. Taken together, our findings suggest that therapeutic concentrations of cisplatin may cause a hybrid type of cell death characterized by concurrent apoptosis and necrosis in the same individual PC12 cell.

  15. Chemically Bonding of Amantadine with Gardenamide A Enhances the Neuroprotective Effects against Corticosterone-Induced Insults in PC12 Cells.

    PubMed

    Zhao, Jiaqiang; Peng, Lizhi; Zheng, Wenhua; Wang, Rikang; Zhang, Lei; Yang, Jian; Chen, Heru

    2015-09-21

    Two amantadine (ATD)-gardenamide A (GA) ligands have been designed and synthesized. The bonding of ATD with GA through a methylene carbonyl brigde (L1) enhances the neuroprotective effect against corticosterone (CORT)-induced impairments in PC12 cells; while the bonding through a succinyl brigde (L2) does not. L1 reduces the level of reactive oxygen species (ROS) and cell apoptosis generated by CORT. It restores CORT-changed cell morphology to a state that is closed to normal PC12 cells. One mechanism of L1 to attenuate CORT-induced cell apoptosis is through the adjustment of both caspase-3 and Bcl-2 proteins. Like GA, both nNOS and eNOS might be involved in the neuroprotective mechanism of L1. All the evidences suggest that L1 may be a potential agent to treat depression.

  16. Interaction of myosin VI and its binding partner DOCK7 plays an important role in NGF-stimulated protrusion formation in PC12 cells.

    PubMed

    Sobczak, Magdalena; Chumak, Vira; Pomorski, Paweł; Wojtera, Emilia; Majewski, Łukasz; Nowak, Jolanta; Yamauchi, Junji; Rędowicz, Maria Jolanta

    2016-07-01

    DOCK7 (dedicator of cytokinesis 7) is a guanidine nucleotide exchange factor (GEF) for Rac1 GTPase that is involved in neuronal polarity and axon generation as well in Schwann cell differentiation and myelination. Recently, we identified DOCK7 as the binding partner of unconventional myosin VI (MVI) in neuronal-lineage PC12 cells and postulated that this interaction could be important in vivo [Majewski et al. (2012) Biochem Cell Biol., 90:565-574]. Herein, we found that MVI-DOCK7 interaction takes also place in other cell lines and demonstrated that MVI cargo domain via its RRL motif binds to DOCK7 C-terminal M2 and DHR2 domains. In MVI knockdown cells, lower Rac1 activity and a decrease of DOCK7 phosphorylation on Tyr1118 were observed, indicating that MVI could contribute to DOCK7 activity. MVI and DOCK7 co-localization was maintained during NGF-stimulated PC12 cell differentiation and observed also in the outgrowths. Also, during differentiation an increase in phosphorylation of DOCK7 as well as of its downstream effector JNK kinase was detected. Interestingly, overexpression of GFP-tagged MVI cargo domain (GFP-GT) impaired protrusion formation indicating that full length protein is important for this process. Moreover, a transient increase in Rac activity observed at 5min of NGF-stimulated differentiation of PC12 cells (overexpressing either GFP or GFP-MVI) was not detected in cells overexpressing the cargo domain. These data indicate that MVI-DOCK7 interaction could have functional implications in the protrusion outgrowth, and full length MVI seems to be important for delivery and maintenance of DOCK7 along the protrusions, and exerting its GEF activity.

  17. Boswellia serrata Protects Against Glutamate-Induced Oxidative Stress and Apoptosis in PC12 and N2a Cells.

    PubMed

    Rajabian, Arezoo; Boroushaki, Mohammad Taher; Hayatdavoudi, Parichehr; Sadeghnia, Hamid Reza

    2016-11-01

    This study was designed to investigate whether the extract from Boswellia serrata oleo-gum resin (BSE) can protect against glutamate-induced oxidative damage and cytotoxicity in PC12 and N2a cell lines. Using a simple and reliable reverse-phase high-performance liquid chromatography (HPLC), the amount of 3-acetyl-11-keto-β-boswellic acid (AKBA) in the BSE was found to be 18.5% w/w. The results confirmed that BSE and AKBA, at concentrations as high as 100 μg/mL or 10 μM, respectively, caused no significant cytotoxicity or apoptotic cell death. Co- and pretreatment with BSE (25-100 μg/mL) or AKBA (5 μM) restored the viability of PC12 and N2a cells under glutamate toxicity (8 mM). Treatment with BSE and AKBA also attenuated the toxic effects of glutamate on intracellular reactive oxygen species, lipid peroxidation, superoxide dismutase activity, and oxidative DNA damage compared with the untreated glutamate-injured cells. Furthermore, BSE and AKBA decreased the apoptotic cell population in the sub-G1 region and the rate of both early and late-stage apoptosis induced by glutamate in the cells. Our data suggest that the protective effects of Boswellia extract and AKBA against glutamate toxicity in PC12 and N2a cells may be mediated through the amelioration of the oxidative stress and the resultant apoptosis.

  18. Effect of Tinospora cordifolia on the reduction of ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells.

    PubMed

    Masuma, Runa; Okuno, Tsutomu; Kabir Choudhuri, Mohammad Shahabuddin; Saito, Takeshi; Kurasaki, Masaaki

    2014-01-01

    The safety of Tinospora cordifolia and its potential to protect against ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells were investigated. To evaluate the safety of T. cordifolia, cell viability and agarose gel electrophoresis were carried out using PC12 cells treated with 0 to 100 μg mL(-1) of methanol extract of T. cordifolia. T. cordifolia extracts did not show cytotoxicity ranging 0 to 100 μg mL(-1). In addition, T. cordifolia extracts significantly increased cell viability at 1 ng, 10 ng and 1 μg mL(-1) concentrations in serum-deprived medium compared to control. To confirm the protective role against UV-induced damage, PC12 cells alone or in the presence of 10 ng, 100 ng, or 1 μg mL(-1) of T. cordifolia extract were exposed to 250, 270 and 290 nm of UV radiation, which corresponded to doses of 120, 150 and 300 mJ cm(-2), respectively. Treatment with T. cordifolia extracts significantly increased the cell survival rate irradiated at 290 nm. In addition, T. cordifolia extracts significantly reduced cyclobutane pyrimidine dimer formation induced by UV irradiation at all wavelengths. In conclusion, T. cordifolia is not toxic and safe for cells. Our findings can support its application as phototherapy in the medical sector.

  19. Identification of miRNAs Involved in the Protective Effect of Sevoflurane Preconditioning Against Hypoxic Injury in PC12 Cells.

    PubMed

    Sun, Yingying; Li, Yuanhai; Liu, Lei; Wang, Yiqiao; Xia, Yingjing; Zhang, Lingli; Ji, Xuewu

    2015-11-01

    The mechanism of sevoflurane preconditioning-induced neuroprotection is poorly understood. This study was aimed at identifying microRNAs (miRNAs) involved in the protective effect of sevoflurane preconditioning against hypoxic injury using the miRCURYTM LNA Array. The screened differentially expressed miRNAs were further validated using qRT-PCR. Finally, after transfection of miRNA (miR-101a or miR-34b) mimics or inhibitor, MTT and flow cytometry assays were used to evaluate cell survival and apoptosis in sevoflurane preconditioning. qRT-PCR confirmed the changes in expression of differentially expressed miRNAs that were screened by the microarray: down-regulation of rno-miR-101a, rno-miR-106b, and rno-miR-294 and up-regulation of rno-miR-883, rno-miR-16, and rno-miR-34b. MiR-101a and miR-34b were the most differentially expressed miRNAs. Sevoflurane preconditioning-inhibited apoptosis and preconditioning-enhanced cell viability of PC12 cells were significantly attenuated by transfection of miR-101a mimetic or miR-34b inhibitors, but were significantly enhanced by transfection of miR-34b mimetic. Therefore, a number of miRNAs, including miR-101a and miR-34b, might play important roles in the neuroprotection induced by sevoflurane preconditioning. Such miRNAs might provide novel targets for preventive and therapeutic strategies against cerebral ischemia-reperfusion injury.

  20. Protective effect of medicinal fungus Xylaria nigripes mycelia extracts against hydrogen peroxide-induced apoptosis in PC12 cells.

    PubMed

    Divate, Rupesh D; Wang, Pei-Ming; Wang, Chiun-Chuang; Chou, Su-Tze; Chang, Chen-Tien; Chung, Yun-Chin

    2017-03-01

    Xylaria nigripes ( XN) is a medicinal fungus that was used traditionally as a diuretic, nerve tonic, and for treating insomnia and trauma. In this study, we elucidated possible mechanisms of neuroprotective effects of XN mycelia extracts. XN mycelia were produced by fermentation. Hot water extract and 70% ethanol extract of XN mycelia were evaluated on hydrogen peroxide (H2O2)-induced apoptosis in PC12, a rat pheochromocytoma cell line. Both XN extracts effectively protected PC12 cells against H2O2-induced cell damage by inhibiting release of lactate dehydrogenase, decreasing DNA damage, restoring mitochondrial membrane potential, and arresting abnormal apoptosis through upregulation of Bcl-2 and downregulation of Bax and caspase 3. Compared to water extract, ethanol extract showed not only greater neuroprotective effects but also a higher antioxidant activity by scavenging DPPH radicals, inhibiting lipid peroxidation, and reducing power. High phenolic content and antioxidant activity may provide the neuroprotective properties of XN ethanol extract.

  1. Overexpression of Annexin A1 Suppresses Pro-Inflammatory Factors in PC12 Cells Induced by 1-Methyl-4-Phenylpyridinium

    PubMed Central

    Kiani-Esfahani, Abbas; Kazemi Sheykhshabani, Sedigheh; Peymani, Maryam; Hashemi, Motahare-Sadat; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

    2016-01-01

    Objective Annexin A1 (ANXA1) is suggested to have anti-inflammatory function. However, the precise function of ANXA1 has remained unclear. In this study, we therefore examined the potency of ANXA1 in regulating reactive oxygen species (ROS) production and suppressing pro-inflammatory responses in PC12 cells induced by 1-methyl-4-phenylpyridinium (MPP+). Materials and Methods In this experimental study, cDNA of ANXA1 was cloned and inserted to the PGL268 pEpi-FGM18F vector to produce a recombinant PGL/ANXA1 vector for transfection into the PC12 cells. ANXA1 transfected cells were then treated with MPP+. Apoptosis and the content of pro-inflammatory factors including ROS, Interlukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) were assessed by flow-cytometry, real-time quantitative polymerase chain reaction (RT-qPCR) and western blot in ANXA1-transfected cells and the data were compared with those obtained from mock and control cells. Results Data revealed that overexpression of ANXA1 is associated with decreased levels of ROS and expression level of IL-6 and iNOS transcripts, and NF-κB protein in MPP+ treated PC12 cells. Conclusion ANXA1 may be considered as an agent for prevention of neurodegenerative or inflammatory conditions. PMID:27540524

  2. Protective effects of statins on L-DOPA neurotoxicity due to the activation of phosphatidylinositol 3-kinase and free radical scavenging in PC12 cell culture.

    PubMed

    Koh, Seong-Ho; Park, Hyun-Hee; Choi, Na-Young; Lee, Kyu-Yong; Kim, Sangjae; Lee, Young Joo; Kim, Hee-Tae

    2011-01-25

    Neurotoxic effects have been suggested for l-3,4-dihydroxyphenylalanine (L-DOPA), while neuroprotective effects have been proposed for statins. We investigated whether certain statins (simvastatin or pitavastatin) could inhibit L-DOPA neurotoxicity. Neuronally-differentiated PC12 (nPC12) cells were treated with L-DOPA and/or statins for 24h, and their viabilities were measured using a cell counting kit, trypan blue staining, and 4',6-diamidino-2-phenylindole (DAPI) staining. Free radical and specific intracellular signaling protein levels were measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and Western blotting, respectively. High concentrations of l-DOPA reduced nPC12 cell viability, but combined treatment with statins restored viability. Treatment with 200 μM L-DOPA increased free radical and hydroxyl radical levels, but combined treatment with 5 μM statins decreased these levels. Survival-related signaling proteins were decreased in nPC12 cells treated with 200 μM L-DOPA, but combined treatment with 5μM statins significantly increased the levels of these proteins. Treatment with 200 μM L-DOPA significantly increased death-related signaling proteins, while combined treatment with 5 μM statins reduced the levels of these proteins. Pretreatment with LY294002, a phosphatidylinositol 3 kinase (PI3K) inhibitor, before combined treatment with statins and L-DOPA almost completely blocked the protective effects of statins. These results indicate that statins reduce L-DOPA neurotoxicity by lowering oxidative stress and by enhancing survival signals and inhibiting death signals via activation of the PI3K pathway.

  3. Protective effects of dibenzocyclooctadiene lignans from Schisandra chinensis against beta-amyloid and homocysteine neurotoxicity in PC12 cells.

    PubMed

    Song, Ju-Xian; Lin, Xiang; Wong, Ricky Ngok-Shun; Sze, Stephen Cho-Wing; Tong, Yao; Shaw, Pang-Chui; Zhang, Yan-Bo

    2011-03-01

    Aggregated beta-amyloid (Aβ) and elevated plasma levels of homocysteine have been implicated as critical factors in the pathogenesis of Alzheimer's disease. The neuroprotective effects and possible mechanism of four structurally similar dibenzocyclooctadiene lignans (namely schisandrin, schisantherin A, schisandrin B and schisandrin C) isolated from the fruit of Schisandra chinensis (Turcz.) Baill. (Schisandraceae) against Aβ₂₅₋₃₅ and homocysteine toxicity in PC12 cells was studied. Exposure of PC12 cells to 0.5 µm Aβ₂₅₋₃₅ caused significant cell death, increased the number of apoptotic cells, elevated reactive oxygen species, increased the levels of the pro-apoptotic protein Bax and caspase-3 activation. All these effects induced by Aβ₂₅₋₃₅ were markedly reversed by schisandrin B and schisandrin C pretreatment, while schisandrin and schisantherin A had no obvious effects. Meanwhile, schisandrin B and schisandrin C reversed homocysteine-induced cytotoxicity. The results indicated that schisandrin B and schisandrin C protected PC12 cells against Aβ toxicity by attenuating ROS production and modulating the apoptotic signal pathway through Bax and caspase-3. Further structure-activity analysis of Schisandra lignans and evaluations of their neuroprotective effects using AD animal models are warranted.

  4. Relative quantification of phospholipid accumulation in the PC12 cell plasma membrane following phospholipid incubation using TOF-SIMS imaging

    PubMed Central

    Lanekoff, Ingela; Sjövall, Peter; Ewing, Andrew G.

    2011-01-01

    Time of flight secondary ion mass spectrometry (TOF-SIMS) imaging has been used to investigate the incorporation of phospholipids into the plasma membrane of PC12 cells after incubation with phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The incubations were done at concentrations previously shown to change the rate of exocytosis in model cell lines. The use of TOF-SIMS in combination with an in situ freeze fracture device enables the acquisition of ion images from the plasma membrane in single PC12 cells. By incubating cells with deuterated phospholipids and acquiring ion images at high mass resolution, specific deuterated fragment ions were used to monitor the incorporation of lipids into the plasma membrane. The concentration of incorporated phospholipids relative to the original concentration of PC was thus determined. The observed relative amounts of phospholipid accumulation in the membrane ranges from 0.5 to 2 percent following 19 hours of incubation with PC at 100 to 300 μM and from 1 to 9 percent following incubation with PE at the same concentrations. Phospholipid accumulation is therefore shown to be dependent on the concentration in the surrounding media. In combination with previous exocytosis results, the present data suggests that very small changes in the plasma membrane phospholipid concentration are sufficient to produce significant effects on important cellular processes, such as exocytosis in PC12 cells. PMID:21563801

  5. Echinacoside Protects against 6-Hydroxydopamine-Induced Mitochondrial Dysfunction and Inflammatory Responses in PC12 Cells via Reducing ROS Production

    PubMed Central

    Wang, Yue-Hua; Xuan, Zhao-Hong; Tian, Shuo; Du, Guan-Hua

    2015-01-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons at the substantia nigra. Mitochondrial dysfunction and inflammatory responses are involved in the mechanism of cell damage in PD. 6-Hydroxydopamine (6-OHDA), a dopamine analog, specifically damages dopaminergic neurons. Echinacoside (ECH) is a phenylethanoid glycoside isolated from the stems of Cistanche salsa, showing a variety of neuroprotective effects in previous studies. The present study was to investigate its effect against 6-OHDA-induced neurotoxicity and possible mechanisms in PC12 cells. The results showed that 6-OHDA reduced cell viability, decreased oxidation-reduction activity, decreased mitochondrial membrane potential, and induced mitochondria-mediated apoptosis compared with untreated PC12 cells. However, echinacoside treatment significantly attenuated these changes induced by 6-OHDA. In addition, echinacoside also could significantly alleviate the inflammatory responses induced by 6-OHDA. Further research showed that echinacoside could reduce 6-OHDA-induced ROS production in PC12 cells. These results suggest that the underlying mechanism of echinacoside against 6-OHDA-induced neurotoxicity may be involve in attenuating mitochondrial dysfunction and inflammatory responses by reducing ROS production. PMID:25788961

  6. Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

    PubMed Central

    Lepsch, Lucilia B; Munhoz, Carolina D; Kawamoto, Elisa M; Yshii, Lidia M; Lima, Larissa S; Curi-Boaventura, Maria F; Salgado, Thais ML; Curi, Rui; Planeta, Cleopatra S; Scavone, Cristoforo

    2009-01-01

    Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells. PMID:19183502

  7. Cocaine induces cell death and activates the transcription nuclear factor kappa-B in PC12 cells.

    PubMed

    Lepsch, Lucilia B; Munhoz, Carolina D; Kawamoto, Elisa M; Yshii, Lidia M; Lima, Larissa S; Curi-Boaventura, Maria F; Salgado, Thais M L; Curi, Rui; Planeta, Cleopatra S; Scavone, Cristoforo

    2009-02-01

    Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-kappaB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-kappaB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-kappaB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-kappaB activation. Inhibition of NF-kappaB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-kappaB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

  8. Chemical constituents from Phyllanthus emblica and the cytoprotective effects on H2O2-induced PC12 cell injuries.

    PubMed

    Zhang, Yao; Zhao, Lijuan; Guo, Xiaojiang; Li, Chao; Li, Haizhen; Lou, Hongxiang; Ren, Dongmei

    2016-09-01

    Two new compounds (1-2), including a bisabolane-type sesquiterpenoid (1), one new diphenyl ether derivative (2), together with 23 known compounds (3-25), were isolated from the fruits of Phyllanthus emblica. Their structures were elucidated by detailed spectroscopic analysis. All the isolated compounds were screened for the DPPH scavenging effects and cytoprotective effects against H2O2 induced PC12 cells injury. Compounds 12-15 showed significant DPPH scavenging effects with the IC50 values in the range of 3.25-4.18 μM. Among these potential antioxidants, compound 14 improved the survival of PC12 cells after H2O2 exposure without showing any cytotoxicity at the tested concentrations.

  9. Neuroprotective effect of D-psicose on 6-hydroxydopamine-induced apoptosis in rat pheochromocytoma (PC12) cells.

    PubMed

    Takata, Maki K; Yamaguchi, Fuminori; Nakanose, Koichi; Watanabe, Yasuo; Hatano, Naoya; Tsukamoto, Ikuko; Nagata, Mitsuhiro; Izumori, Ken; Tokuda, Masaaki

    2005-11-01

    We evaluated the neuroprotective effects of D-psicose, one of the rare sugars, on 6-hydroxydopamine (6-OHDA)-induced apoptosis in catecholaminergic PC12 cells, the in vitro model of Parkinson's disease (PD). Apoptotic characteristics of PC12 cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) assay. The results showed that D-psicose at a concentration of 50 mM, exerted significant protective effects against the 6-OHDA (200 muM)-induced PC12 cell apoptosis, while other sugars had little or no protective effects. We have observed a significant increase in the level of intracellular glutathione after 24 h in 6-OHDA (200 muM) treated cells, while a decrease in the level was observed at 3 h and 6 h. Also, a synergistic exposure to D-psicose and 6-OHDA for 24 h showed a significant increase in intracellular glutathione level. Therefore, these results suggest that D-psicose may play a potential role as a neuroprotective agent in the treatment of neurodegenerative diseases by inducing an up-regulation of intracellular glutathione.

  10. Deep hypothermia-enhanced autophagy protects PC12 cells against oxygen glucose deprivation via a mitochondrial pathway.

    PubMed

    Tang, Dang; Wang, Cheng; Gao, Yongjun; Pu, Jun; Long, Jiang; Xu, Wei

    2016-10-06

    Deep hypothermia is known for its organ-preservation properties, which is introduced into surgical operations on the brain and heart, providing both safety in stopping circulation as well as an attractive bloodless operative field. However, the molecular mechanisms have not been clearly identified. This study was undertaken to determine the influence of deep hypothermia on neural apoptosis and the potential mechanism of these effects in PC12 cells following oxygen-glucose deprivation. Deep hypothermia (18°C) was given to PC12 cells while the model of oxygen-glucose deprivation (OGD) induction for 1h. After 24h of reperfusion, the results showed that deep hypothermia decreased the neural apoptosis, and significantly suppressed overexpression of Bax, CytC, Caspase 3, Caspase 9 and cleaved PARP-1, and inhibited the reduction of Bcl-2 expression. While deep hypothermia increased the LC3II/LC3I and Beclin 1, an autophagy marker, which can be inhibited by 3-methyladenine (3-MA), indicating that deep hypothermia-enhanced autophagy ameliorated apoptotic cell death in PC12 cells subjected to OGD. Based on these findings we propose that deep hypothermia protects against neural apoptosis after the induction of OGD by attenuating the mitochondrial apoptosis pathway, moreover, the mechanism of these antiapoptosis effects is related to the enhancement of autophagy, which autophagy might provide a means of neuroprotection against OGD.

  11. Nitric oxide enhances increase in cytosolic Ca(2+) and promotes nicotine-triggered MAPK pathway in PC12 cells.

    PubMed

    Kajiwara, Aya; Tsuchiya, Yukihiro; Takata, Tsuyoshi; Nyunoya, Mayumi; Nozaki, Naohito; Ihara, Hideshi; Watanabe, Yasuo

    2013-11-01

    The purpose of this study was to investigate the roles of neuronal nitric oxide synthase (nNOS), Ca(2+)/calmodulin (CaM)-dependent protein kinases (CaMKs), and protein kinase C (PKC) in nicotine-induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) activation. Treatment with nicotine stimulated ERK1/2 and p38 MAPK phosphorylation in the PC12 cells expressing nNOS (NPC12 cells) as compared with that in control PC12 cells. An inhibitor of L-type voltage-sensitive Ca(2+) channel suppressed the nicotine-induced phosphorylation of p38 MAPK. The inhibition of CaMK-kinase, the upstream activator of CaMKI and CaMKIV, did not inhibit the enhanced their phosphorylation. ERK1/2 phosphorylation was attenuated by inhibitors of p38 MAPK, PKC, and MAPK-kinase 1/2, indicating the involvement of these protein kinases upstream of ERK1/2. Furthermore, we found that nNOS expression enhances the nicotine-induced increase in the intracellular concentration of Ca(2+), using the Ca(2+)-sensitive fluorescent probe Fura2. These data suggest that NO promotes nicotine-triggered Ca(2+) transient in PC12 cells to activate possibly CaMKII, leading to sequential phosphorylation of p38 MAPK and ERK1/2.

  12. Quercetin and sesamin protect neuronal PC12 cells from high-glucose-induced oxidation, nitrosative stress, and apoptosis.

    PubMed

    Bournival, Julie; Francoeur, Marc-André; Renaud, Justine; Martinoli, Maria-Grazia

    2012-06-01

    Complications of diabetes are now well-known to affect sensory, motor, and autonomic nerves. Diabetes is also thought to be involved in neurodegenerative processes characteristic of several neurodegenerative diseases. Indeed, it has been acknowledged recently that hyperglycemia-induced oxidative stress contributes to numerous cellular reactions typical of central nervous system deterioration. The goal of the present study was to evaluate the effects of the polyphenol quercetin and the lignan sesamin on high-glucose (HG)-induced oxidative damage in an in vitro model of dopaminergic neurons, neuronal PC12 cells. When incubated with HG (13.5 mg/mL), neuronal PC12 cells showed a significant increase of cellular death. Our results revealed that quercetin and sesamin defend neuronal PC12 cells from HG-induced cellular demise. An elevated level of reactive oxygen and nitrogen species is a consequence of improved oxidative stress after HG administration, and we demonstrated that this production diminishes with quercetin and sesamin treatment. We also found that quercetin and sesamin elicited an increment of superoxide dismutase activity. DNA fragmentation, Bax/Bcl-2 ratio, nuclear translocation of apoptosis-inducing factor, as well as poly(adenosine diphosphate [ADP]-ribose) polymerase cleavage were significantly reduced by quercetin and sesamin administration, affirming their antiapoptotic features. Also, HG treatment impacted caspase-3 cleavage, supporting caspase-3-dependent pathways as mechanisms of apoptotic death. Our results indicate a powerful role for these natural dietary compounds and emphasize preventive or complementary nutritional strategies for diabetes control.

  13. Induction of Serpinb1a by PACAP or NGF is required for PC12 cells survival after serum withdrawal

    PubMed Central

    Seaborn, Tommy; Ravni, Aurélia; Au, Ruby; Chow, Bill K.C.; Fournier, Alain; Wurtz, Olivier; Vaudry, Hubert; Eiden, Lee E.; Vaudry, David

    2014-01-01

    PC12 cells are used to study the signaling mechanisms underlying the neurotrophic and neuroprotective activities of pituitary adenylate cyclase-activating polypeptide (PACAP) and nerve growth factor (NGF). Previous microarray experiments indicated that serpinb1a was the most induced gene after 6 h of treatment with PACAP or NGF. The present study confirmed that serpinb1a is strongly activated by PACAP and NGF in a time-dependent manner with a maximum induction (~50-fold over control) observed after 6 h of treatment. Co-incubation with PACAP and NGF resulted in a synergistic up-regulation of serpinb1a expression (200-fold over control), suggesting that PACAP and NGF act through complementary mechanisms. Consistently, PACAP-induced serpinb1a expression was not blocked by TrkA receptor inhibition. Nevertheless, the stimulation of serpinb1a expression by PACAP and NGF was significantly reduced in the presence of ERK, calcineurin, PKA, p38 and PI3K inhibitors, indicating that the two trophic factors share some common pathways in the regulation of serpinb1a. Finally, functional investigations conducted with siRNA revealed that serpinb1a is not involved in the effects of PACAP and NGF on PC12 cell neuritogenesis, proliferation or body volume but mediates their ability to block caspase-3/7 activity and to promote PC12 cell survival. PMID:24899316

  14. Colistin-induced apoptosis in PC12 cells: involvement of the mitochondrial apoptotic and death receptor pathways.

    PubMed

    Jiang, Hong; Li, Jichang; Zhou, Tiezhong; Wang, Chunhua; Zhang, Hua; Wang, Hongjun

    2014-05-01

    Colistin, a cyclic cationic polypeptide antibiotic that is used to treat infections, may cause neurotoxicity. However, whether colistin can induce apoptosis and the precise mechanism of apoptosis involved in PC12 cells remains to be determined. The aim of the present study was to determine reactive oxygen species (ROS) level and DNA damage, as well as apoptotic factors such as p53, cytochrome c, Bax, Bcl-2, Fas, Fas-L and caspase family via western blotting in PC12 cells treated with colistin sulfate. The results showed that colistin sulfate increased ROS levels significantly. An increase of ROS levels induces the release of cytochrome c and DNA damage. DNA damage can activate p53, which leads to the upregulation of Bax and downregulation of Bcl-2. The imbalance of Bax/Bcl-2 promotes additional release of cytochrome c. The release of cytochrome c contributes to the activation of caspase-9 and the subsequent activation of caspase-3. An increase of Fas and Fas-L induced the activation of caspase-8 leading to the activation of caspases-3, the latter induces apoptosis. Therefore, these results demonstrate that the apoptotic pathway of colistin-induced apoptosis in PC12 cells is involved in both the mitochondrial and death receptor pathway.

  15. Antineurodegenerative effect of phenolic extracts and caffeic acid derivatives in romaine lettuce on neuron-like PC-12 cells.

    PubMed

    Im, Sung-Eun; Yoon, Hyungeun; Nam, Tae-Gyu; Heo, Ho Jin; Lee, Chang Yong; Kim, Dae-Ok

    2010-08-01

    In recent decades, romaine lettuce has been one of the fastest growing vegetables with respect to its consumption and production. An understanding is needed of the effect of major phenolic phytochemicals from romaine lettuce on biological protection for neuron-like PC-12 cells. Phenolics in fresh romaine lettuce were extracted, and then its total phenolics and total antioxidant capacity were measured spectrophotometrically. Neuroprotective effects of phenolic extract of romaine lettuce and its pure caffeic acid derivatives (caffeic, chicoric, chlorogenic, and isochlorogenic acids) in PC-12 cells were evaluated using two different in vitro methods: lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assays. Total phenolics and total antioxidant capacity of 100 g of fresh romaine lettuce averaged 22.7 mg of gallic acid equivalents and 31.0 mg of vitamin C equivalents, respectively. The phenolic extract of romaine lettuce protected PC-12 cells against oxidative stress caused by H(2)O(2) in a dose-dependent manner. Isochlorogenic acid, one of the phenolics in romaine lettuce, showed stronger neuroprotection than the other three caffeic acid derivatives also found in the lettuce. Although romaine lettuce had lower levels of phenolics and antioxidant capacity compared to other common vegetables, its contribution to total antioxidant capacity and antineurodegenerative effect in human diets would be higher because of higher amounts of its daily per capita consumption compared to other common vegetables.

  16. Nerve growth factor enhances the CRE-dependent transcriptional activity activated by nobiletin in PC12 cells.

    PubMed

    Takito, Jiro; Kimura, Junko; Kajima, Koji; Uozumi, Nobuyuki; Watanabe, Makoto; Yokosuka, Akihito; Mimaki, Yoshihiro; Nakamura, Masanori; Ohizumi, Yasushi

    2016-07-01

    Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells.

  17. BDNF-TrkB Pathway Mediates Neuroprotection of Hydrogen Sulfide against Formaldehyde-Induced Toxicity to PC12 Cells

    PubMed Central

    Gao, Sheng-Lan; Tian, Ying; Wang, Chun-Yan; Wang, Li; Gu, Hong-Feng; Tang, Xiao-Qing

    2015-01-01

    Formaldehyde (FA) is a common environmental contaminant that has toxic effects on the central nervous system (CNS). Our previous data demonstrated that hydrogen sulfide (H2S), the third endogenous gaseous mediator, has protective effects against FA-induced neurotoxicity. As is known to all, Brain-derived neurotropic factor (BDNF), a member of the neurotrophin gene family, mediates its neuroprotective properties via various intracellular signaling pathways triggered by activating the tyrosine kinase receptor B (TrkB). Intriguingly, our previous data have illustrated the upregulatory role of H2S on BDNF protein expression in the hippocampus of rats. Therefore, in this study, we hypothesized that H2S provides neuroprotection against FA toxicity by regulating BDNF-TrkB pathway. In the present study, we found that NaHS, a donor of H2S, upregulated the level of BDNF protein in PC12 cells, and significantly rescued FA-induced downregulation of BDNF levels. Furthermore, we found that pretreatment of PC12 cells with K252a, an inhibitor of the BDNF receptor TrkB, markedly reversed the inhibition of NaHS on FA-induced cytotoxicity and ablated the protective effects of NaHS on FA-induced oxidative stress, including the accumulation of intracellular reactive oxygen species (ROS), 4-hydroxy-2-trans-nonenal (4-HNE), and malondialdehyde (MDA). We also showed that K252a abolished the inhibition of NaHS on FA-induced apoptosis, as well as the activation of caspase-3 in PC12 cells. In addition, K252a reversed the protection of H2S against FA-induced downregulation of Bcl-2 protein expression and upregulation of Bax protein expression in PC12 cells. These data indicate that the BDNF-TrkB pathway mediates the neuroprotection of H2S against FA-induced cytotoxicity, oxidative stress and apoptosis in PC12 cells. These findings provide a novel mechanism underlying the protection of H2S against FA-induced neurotoxicity. PMID:25749582

  18. Recombinant human TAT-OP1 to enhance NGF neurogenic potential: preliminary studies on PC12 cells.

    PubMed

    Di Liddo, R; Grandi, C; Venturini, M; Dalzoppo, D; Negro, A; Conconi, M T; Parnigotto, P P

    2010-11-01

    Osteogenic protein 1 (OP1), also known as bone morphogenic protein-7 (BMP7), is a multifunctional cytokine with demonstrated neurogenic potential. As the recombinant OP1 (rhOP1) was shown to provide axonal guidance cues and to prevent the reduction of dendritic growth in the injury-induced cortical cultures, it was suggested that an in vivo efficient rhOP1 delivery could enhance neurite growth and functional reconnectivity in the damaged brain. In the present work, we engineered a chimeric molecule in which rhBMP7 was fused to a protein transduction domain derived from HIV-1 TAT protein to deliver the denatured recombinant BMP7 into cells and obtain its chaperone-mediated folding, circumventing the expensive and not much efficient in vitro refolding procedures. When tested on rat PC12 cells, a widely used in vitro neurogenic differentiation model, the resulting fusion protein (rhTAT-OP1) demonstrated to enter fastly into the cells, lose HIV-TAT sequence and interact with membrane receptors activating BMP pathway by SMAD 1/5/8 phosphorylation. In comparison with nerve growth factor (NGF) and BMP7, it proved itself effective to induce the formation of more organized H and M neurofilaments. Moreover, if used in combination with NGF, it stimulated a significant (P < 0.05) and more precocious dendritic outgrowth with respect to NGF alone. These results indicate that rhTAT-OP1 fused with TAT transduction domain shows neurogenic activity and may be a promising enhancer factor in NGF-based therapies.

  19. Nanogrooved surface-patterns induce cellular organization and axonal outgrowth in neuron-like PC12-cells.

    PubMed

    Klymov, Alexey; Rodrigues Neves, Charlotte T; te Riet, Joost; Agterberg, Martijn J H; Mylanus, Emmanuel A M; Snik, Ad F M; Jansen, John A; Walboomers, X Frank

    2015-02-01

    Modulation of a materials surface topography can be used to steer various aspects of adherent cell behaviour, such as cell directional organization. Especially nanometric sized topographies, featuring sizes similar to for instance the axons of the spiral ganglion cells, are interesting for such purpose. Here, we utilized nanosized grooves in the range of 75-500 nm, depth of 30-150 nm, and pitches between 150 nm and 1000 nm for cell culture of neuron-like PC12 cells. The organizational behaviour was evaluated after 7 days of culture by bright field and scanning electron microscopy. Nanotopographies were shown to induce aligned cell-body/axon orientation and an increased axonal outgrowth. Our findings suggest that a threshold for cell body alignment of neuronal cells exists on grooved topographies with a groove width of 130 nm, depth of 70 nm and pitch of 300 nm, while axon alignment can already be induced by grooves with 135 nm width, 52 nm depth and 200 nm pitch. However, no threshold has been found for axonal outgrowth, as all of the used patterns increased outgrowth of PC12-axons. In conclusion, surface nanopatterns have the potential to be utilized as an electrode modification for a stronger separation of cells, and can be used to direct cells towards the electrode contacts of cochlear implants.

  20. Nerve growth factor-induced neurite sprouting in PC12 cells involves sigma-1 receptors: implications for antidepressants.

    PubMed

    Takebayashi, Minoru; Hayashi, Teruo; Su, Tsung-Ping

    2002-12-01

    One theory concerning the action of antidepressants relates to the drugs' ability to induce an adaptive plasticity in neurons such as neurite sprouting. Certain antidepressants are known to bind to sigma-1 receptors (Sig-1R) with high affinity. Sig-1R are dynamic endoplasmic reticulum proteins that are highly concentrated at the tip of growth cones in cultured cells. We therefore tested the hypotheses that Sig-1R might participate in the neurite sprouting and that antidepressants with Sig-1R affinity may promote the neuronal sprouting via Sig-1R. The prototypic Sig-1R agonist (+)-pentazocine [(+)PTZ], as well as the Sig-1R-active antidepressants imipramine and fluvoxamine, although ineffective by themselves, were found to enhance the nerve growth factor (NGF)-induced neurite sprouting in PC12 cells in a dose-dependent manner. A Sig-1R antagonist N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE100) blocked the enhancements caused by these Sig-1R agonists. In separate experiments, we found that NGF dose and time dependently increased Sig-1R in PC12 cells. Chronic treatment of cells with (+)PTZ, imipramine, or fluvoxamine also increased Sig-1R. These latter results suggested that NGF induces the neurite sprouting by increasing Sig-1R. Indeed, the overexpression of Sig-1R per se in PC12 cells enhanced the NGF-induced neurite sprouting. Furthermore, antisense deoxyoligonucleotides directed against Sig-1R attenuated the NGF-induced neurite sprouting. Thus, when taken together, our results indicate that Sig-1R play an important role in the NGF-induced neurite sprouting and that certain antidepressants may facilitate neuronal sprouting in the brain via Sig-1R.

  1. Interaction of single and multi wall carbon nanotubes with the biological systems: tau protein and PC12 cells as targets

    PubMed Central

    Zeinabad, Hojjat Alizadeh; Zarrabian, Alireza; Saboury, Ali Akbar; Alizadeh, Ali Mohammad; Falahati, Mojtaba

    2016-01-01

    Subtle changes in the structure of nanoparticles influence their surface tension and corresponding interaction with cells and proteins. Here, the interaction of the single wall carbon nanotube (SWCNT) and multiwall carbon nanotube (MWCNT) with different surface tension with tau protein was evaluated using a variety of techniques including far and near circular dichroism, fluorescence spectroscopy, dynamic light scattering, Zeta potential, and TEM evaluation. Also the cytotoxicity of SWCNT and MWCNT on the PC12 cell line as a model of nervous system cell line was investigated by the MTT, LDH, acridine orange/ethidium bromide staining, flow cytometry, caspase 3 activity, cell and membrane potential assays. It was observed that SWCNT induced more structural changes of tau protein relative to MWCNT/tau protein interaction. It was also revealed that SWCNT and MWCNT impaired the viability and complexity of PC12 cells in different modes of cytotoxicity. Analysis of cellular outcomes indicated that MWCNT in comparison with SWCNT resulted in induction of necrotic modes of cell death, whereas apoptotic modes of cell death were activated in SWCNT-incubated cells. Together these findings suggest that surface tension may be used to determine how nanoparticle structure affects neurotoxicity and protein conformational changes. PMID:27216374

  2. Phlorofucofuroeckol Improves Glutamate-Induced Neurotoxicity through Modulation of Oxidative Stress-Mediated Mitochondrial Dysfunction in PC12 Cells

    PubMed Central

    Shin, Sun-Ae; Bak, Dong-Ho; Lee, Jae Won; Lee, Kyung Bok; Yoo, Yung Choon; Kim, Do-Kyung; Lee, Bong Ho; Kim, Dong Woon; Lee, Jina; Jo, Eun-Kyeong

    2016-01-01

    Stroke is a complex neurodegenerative disorder with a clinically high prevalence and mortality. Despite many efforts to protect against ischemic stroke, its incidence and related permanent disabilities continue to increase. In this study, we found that pretreatment with phlorofucofuroeckol (PFF), isolated from brown algae species, significantly increased cell viability in glutamate-stimulated PC12 cells. Additionally, glutamate-stimulated cells showed irregular morphology, but PFF pretreatment resulted in improved cell morphology, which resembled that in cells cultured under normal conditions. We further showed that PFF pretreatment effectively inhibited glutamate-induced apoptotic cell death in a caspase-dependent manner. Reactive oxygen species (ROS) induced by oxidative stress are closely associated with ischemia-induced neurological diseases. Exposure of PC12 cells to glutamate induced abundant production of intracellular ROS and mitochondrial dysfunction, which was attenuated by PFF in a dose-dependent manner. In vivo studies revealed that PFF-mediated prevention was achieved predominantly through inhibition of apoptosis and mitochondrial ROS generation. Taken together, these results suggest the possibility of PFF as a neuroprotective agent in ischemic stroke. PMID:27669570

  3. Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity

    NASA Astrophysics Data System (ADS)

    Leach, Jennie B.; Brown, Xin Q.; Jacot, Jeffrey G.; Di Milla, Paul A.; Wong, Joyce Y.

    2007-06-01

    Rationally designed matrices for nerve tissue engineering and encapsulated cell therapies critically rely on a comprehensive understanding of neural response to biochemical as well as biophysical cues. Whereas biochemical cues are established mediators of neuronal behavior (e.g., outgrowth), physical cues such as substrate stiffness have only recently been recognized to influence cell behavior. In this work, we examine the response of PC12 neurites to substrate stiffness. We quantified and controlled fibronectin density on the substrates and measured multiple neurite behaviors (e.g., growth, branching, neurites per cell, per cent cells expressing neurites) in a large sample population. We found that PC12 neurons display a threshold response to substrate stiffness. On the softest substrates tested (shear modulus ~10 Pa), neurites were relatively few, short in length and unbranched. On stiffer substrates (shear modulus ~102-104 Pa), neurites were longer and more branched and a greater percentage of cells expressed neurites; significant differences in these measures were not found on substrates with a shear modulus >102 Pa. Based on these data and comparisons with published neurobiology and neuroengineering reports of neurite mechanotransduction, we hypothesize that results from studies of neuronal response to compliant substrates are cell-type dependent and sensitive to ligand density, sample size and the range of stiffness investigated.

  4. Evaluation of In-Situ Magnetic Signals from Iron Oxide Nanoparticle-Labeled PC12 Cells by Atomic Force Microscopy.

    PubMed

    Wang, Lijun; Min, Yue; Wang, Zhigang; Riggio, Cristina; Calatayud, M Pilar; Pinkernelle, Josephine; Raffa, Vittoria; Goya, Gerardo F; Keilhoff, Gerburg; Cuschieri, Alfred

    2015-03-01

    The magnetic signals from magnetite nanoparticle-labeled PC12 cells were assessed by magnetic force microscopy by deploying a localized external magnetic field to magnetize the nanoparticles and the magnetic tip simultaneously so that the interaction between the tip and PC12 cell-associated Fe3O4 nanoparticles could be detected at lift heights (the distance between the tip and the sample) larger than 100 nm. The use of large lift heights during the raster scanning of the probe eliminates the non-magnetic interference from the complex and rugged cell surface and yet maintains the sufficient sensitivity for magnetic detection. The magnetic signals of the cell-bound nanoparticles were semi-quantified by analyzing cell surface roughness upon three-dimensional reconstruction generated by the phase shift of the cantilever oscillation. The obtained data can be used for the evaluation of the overall cellular magnetization as well as the maximum magnetic forces from magnetic nanoparticle-labeled cells which is crucial for the biomedical application of these nanomaterials.

  5. Tris (1,3-dichloro-2-propyl) phosphate induces toxicity by stimulating CaMK2 in PC12 cells.

    PubMed

    Li, Chaonan; Li, Li; Lin, Bencheng; Fang, Yanjun; Yang, Honglian; Liu, Huanliang; Xi, Zhuge

    2017-02-09

    Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is one of the widely used organophosphorus flame retardants (OPFRs), which are regarded as suitable substitutes for brominated flame retardants (BFRs). Previously, we have validated the toxicity of TDCIPP in PC12 cells owing to the induced alterations in GAP43, NF-H, CaMK2a/2b, and tubulin α/β proteins; however, limited information is currently available on the toxicity and mechanism of TDCIPP. In the present study, cytotoxicity effects were evaluated by exposing PC12 cells to different concentrations of TDCIPP (0-50 μM) for 4 days. To explore the possible mechanisms through which cytotoxicity is induced, changes in intracellular [Ca(2+) ]i levels and the activation of calmodulin dependent protein kinase 2 (CaMK2), c-Jun N-terminal kinase (JNK), extracellular regulated protein kinases (ERK1/2), and p38 mitogen-activated protein kinases (MAPK) pathways were evaluated. Furthermore, PC12 cells were pretreated with CaMK2 inhibitor KN93 to investigate the relationship between TDCIPP-induced phosphorylation of CaMK2 and activation of JNK, ERK1/2, and p38 MAPK pathways. Our results indicate that TDCIPP-induced toxicity might be associated with the overload of [Ca(2+) ]i levels, increased phosphorylation of CaMK2, and activation of the JNK, ERK1/2, and p38 MAPK pathways, the lattermost of which was further demonstrated to be partially elicited by the CaMK2 phosphorylation.

  6. Microchip-based integration of cell immobilization, electrophoresis, post-column derivatization, and fluorescence detection for monitoring the release of dopamine from PC 12 cells.

    PubMed

    Li, Michelle W; Martin, R Scott

    2008-10-01

    In this paper, we describe the fabrication and evaluation of a multilayer microchip device that can be used to quantitatively measure the amount of catecholamines released from PC 12 cells immobilized within the same device. This approach allows immobilized cells to be stimulated on-chip and, through rapid actuation of integrated microvalves, the products released from the cells are repeatedly injected into the electrophoresis portion of the microchip, where the analytes are separated based upon mass and charge and detected through post-column derivatization and fluorescence detection. Following optimization of the post-column derivatization detection scheme (using naphthalene-2,3-dicarboxaldehyde and 2-beta-mercaptoethanol), off-chip cell stimulation experiments were performed to demonstrate the ability of this device to detect dopamine from a population of PC 12 cells. The final 3-dimensional device that integrates an immobilized PC 12 cell reactor with the bilayer continuous flow sampling/electrophoresis microchip was used to continuously monitor the on-chip stimulated release of dopamine from PC 12 cells. Similar dopamine release was seen when stimulating on-chip versus off-chip yet the on-chip immobilization studies could be carried out with 500 times fewer cells in a much reduced volume. While this paper is focused on PC 12 cells and neurotransmitter analysis, the final device is a general analytical tool that is amenable to the immobilization of a variety of cell lines and analysis of various released analytes by electrophoretic means.

  7. Acetylated Chitosan Oligosaccharides Act as Antagonists against Glutamate-Induced PC12 Cell Death via Bcl-2/Bax Signal Pathway

    PubMed Central

    Hao, Cui; Gao, Lixia; Zhang, Yiran; Wang, Wei; Yu, Guangli; Guan, Huashi; Zhang, Lijuan; Li, Chunxia

    2015-01-01

    Chitosan oligosaccharides (COSs), depolymerized products of chitosan composed of β-(1→4) d-glucosamine units, have broad range of biological activities such as antitumour, antifungal, and antioxidant activities. In this study, peracetylated chitosan oligosaccharides (PACOs) and N-acetylated chitosan oligosaccharides (NACOs) were prepared from the COSs by chemcal modification. The structures of these monomers were identified using NMR and ESI-MS spectra. Their antagonist effects against glutamate-induced PC12 cell death were investigated. The results showed that pretreatment of PC12 cells with the PACOs markedly inhibited glutamate-induced cell death in a concentration-dependent manner. The PACOs were better glutamate antagonists compared to the COSs and the NACOs, suggesting the peracetylation is essential for the neuroprotective effects of chitosan oligosaccharides. In addition, the PACOs pretreatment significantly reduced lactate dehydrogenase release and reactive oxygen species production. It also attenuated the loss of mitochondrial membrane potential. Further studies indicated that the PACOs inhibited glutamate-induced cell death by preventing apoptosis through depressing the elevation of Bax/Bcl-2 ratio and caspase-3 activation. These results suggest that PACOs might be promising antagonists against glutamate-induced neural cell death. PMID:25775423

  8. Protective Effect of Psoralea corylifolia L. Seed Extract against Palmitate-Induced Neuronal Apoptosis in PC12 Cells

    PubMed Central

    Lee, Yunkyoung

    2016-01-01

    The extract of Psoralea corylifolia seeds (PCE) has been widely used as a herbal medicine because of its beneficial effect on human health. In this study, we investigated the protective effects and molecular mechanisms of PCE on palmitate- (PA-) induced toxicity in PC12 cells, a neuron-like cell line. PCE significantly increased cell viability in PA-treated PC12 cells and showed antiapoptotic effects, as evidenced by decreased expression of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase, and bax protein as well as increased expression of bcl-2 protein. In addition, PCE treatment reduced PA-induced reactive oxygen species production and upregulated mRNA levels of antioxidant genes such as nuclear factor (erythroid-derived 2)-like 2 and heme oxygenase 1. Moreover, PCE treatment recovered the expression of autophagy marker genes such as beclin-1 and p62, which was decreased by PA treatment. Treatment with isopsoralen, one of the major components of PCE extract, also recovered the expression of autophagy marker genes and reduced PA-induced apoptosis. In conclusion, PCE exerts protective effects against lipotoxicity via its antioxidant function, and this effect is mediated by activation of autophagy. PCE might be a potential pharmacological agent to protect against neuronal cell injury caused by oxidative stress or lipotoxicity. PMID:27843479

  9. Aripiprazole increases NAD(P)H-quinone oxidoreductase-1 and heme oxygenase-1 in PC12 cells.

    PubMed

    Kaneko, Yoko S; Takayanagi, Takeshi; Nagasaki, Hiroshi; Kodani, Yu; Nakashima, Akira; Mori, Keiji; Suzuki, Atsushi; Itoh, Mitsuyasu; Kondo, Kazunao; Nagatsu, Toshiharu; Ota, Miyuki; Ota, Akira

    2015-06-01

    We previously showed that aripiprazole increases intracellular NADPH and glucose-6-phosphate dehydrogenase mRNA in PC12 cells. Aripiprazole presumably activates a system that concurrently detoxifies reactive oxygen species and replenishes NADPH. Nrf2, a master transcriptional regulator of redox homeostasis genes, also activates the pentose phosphate pathway, including NADPH production. Therefore, our aim was to determine whether aripiprazole activates Nrf2 in PC12 cells. Aripiprazole increased mRNA expression of Nrf2-dependent genes (NAD(P)H-quinone oxidoreductase-1, Nqo1; heme oxygenase-1, HO1; and glutamate-cysteine ligase catalytic subunit) and protein expression of Nqo1 and HO1 in these cells (p < 0.05). To maintain increased Nrf2 activity, it is necessary to inhibit Nrf2 degradation; this is done by causing Nrf2 to dissociate from Keap1 or β-TrCP. However, in aripiprazole-treated cells, the relative amount of Nrf2 anchored to Keap1 or β-TrCP was unaffected and Nrf2 in the nuclear fraction decreased (p < 0.05). Aripiprazole did not affect phosphorylation of Nrf2 at Ser40 and decreased the relative amount of acetylated Nrf2 (p < 0.05). The increase in Nqo1 and HO1 in aripiprazole-treated cells cannot be explained by the canonical Nrf2-degrading pathways. Further experiments are needed to determine the biochemical mechanisms underlying the aripiprazole-induced increase in these enzymes.

  10. Protective effect of naringin against the LPS-induced apoptosis of PC12 cells: Implications for the treatment of neurodegenerative disorders

    PubMed Central

    Wang, Hui; Xu, You Song; Wang, Miao Lin; Cheng, Chao; Bian, Rui; Yuan, Hao; Wang, Yi; Guo, Ting; Zhu, Lin Lin; Zhou, Hang

    2017-01-01

    Several studies have demonstrated that increased apoptosis plays an essential role in neurodegenerative disorders. It has been demonstrated that lipopolysaccharide (LPS) induces apoptosis largely through the production of intracellular reactive oxygen species (ROS) and inflammatory mediators. In this study, we investigated the potential protective mechanisms of naringin (Nar), a pummelo peel extract, on LPS-induced PC12 cell apoptosis. Nar pre-conditioning prior to stimulation with LPS for 18 h was a prerequisite for evaluating PC12 cell viability and the protective mechanisms of Nar. Nar significantly improved cell survival in a time- and concentration-dependent manner. On the one hand, Nar downregulated cytochrome P450 2E1 (CYP2E1), inhibited the release of ROS, mitigated the stimulation of oxidative stress, and rectified the antioxidant protein contents of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), superoxide dismutase (SOD)2 and glutathione synthetase (GSS). On the other hand, Nar down-regulated inflammatory gene and protein expression, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, HMGB1, high mobility group box 1 protein (HMGB1), cyclo-oxygenase-2 (COX-2), the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-TNF receptor-associated factor 6 (TRAF6) path way and downstream mitogen activated protein kinase (MAPK) phosphorylation, activator protein transcription factor-1 (AP-1) and nuclear factor (NF)-κB. Moroever, Nar markedly attenuated the cytochrome c shift from the mitochondria to the cytosol and regulated caspase-3-related protein expression. To the best of our knowledge, this is the first study to report the antioxidant, anti-inflammatory and anti-apoptotic effects of Nar in neuronal-like PC12 cells. These results suggest that Nar can be utilized as a potential drug for the treatment of neurodegenerative disorders. PMID:28260042

  11. Tetrahydroxystilbene glucoside attenuates MPP+-induced apoptosis in PC12 cells by inhibiting ROS generation and modulating JNK activation.

    PubMed

    Li, Xiaobing; Li, Yan; Chen, Jianzong; Sun, Jing; Li, Xiaofeng; Sun, Xin; Kang, Xiaogang

    2010-10-08

    It is known that oxidative stress plays a major role in the progression of Parkinson's disease (PD). Previous studies have suggested that 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside (TSG), an active component extracted from a traditional Chinese herb Polygonum multiflorum Thunb., has significant antioxidant and free radical-scavenging activities. This is the first study that investigated the protective effects of TSG against MPP(+)-induced apoptosis in PC12 cells and determined the underlying mechanism. The results showed that incubation of PC12 cells with TSG before exposing them to MPP(+) could significantly decrease cell viability loss and reverse cell apoptosis in a dose-dependent manner. The anti-apoptotic effects of TSG were probably mediated via the inhibition of ROS generation and modulation of JNK activation because TSG blocked ROS increase and JNK phosphorylation induced by MPP(+). Taken together, these results indicated that TSG may provide a useful therapeutic strategy for the treatment of neurodegenerative diseases such as PD.

  12. Chitosan attenuates dibutyltin-induced apoptosis in PC12 cells through inhibition of the mitochondria-dependent pathway.

    PubMed

    Wang, Xiaorui; Miao, Junqiu; Yan, Chaoqun; Ge, Rui; Liang, Taigang; Liu, Enli; Li, Qingshan

    2016-10-20

    Dibutyltin (DBT) which was widely used as biocide and plastic stabilizer has been described as a potent neurotoxicant. Chitosan (CS), a natural nontoxic biopolymer, possesses a variety of biological activities including antibacterial, antifungal, free radical scavenging and neuroprotective activities. The present study was undertaken to investigate the protective effects of CS against DBT-induced apoptosis in rat pheochromocytoma (PC12) cells and the underlying mechanisms in vitro. Our results demonstrated that pretreatment with CS significantly increased the cell viability and decreased lactate dehydrogenase (LDH) release induced by DBT in a dose-dependent manner. Meanwhile, DBT-induced cell apoptosis, mitochondrial membrane potential (MMP) disruption, and generation of intracellular reactive oxygen species (ROS) were attenuated by CS. Real-time PCR assay showed that DBT markedly enhanced the mRNA levels of Bax, Bad, cytochrome-c and Apaf-1, reduced the Bcl-2 and Bcl-xL mRNA levels, while these genes expression alteration could be partially reversed by CS treatment. Furthermore, CS also inhibited the DBT-inducted activation of caspase-9, and -3 at mRNA and protein expression levels. Taken together, these results suggested that CS could protect the PC12 cells from apoptosis induced by DBT through inhibition of the mitochondria-dependent pathway.

  13. Epidermal growth factor prevents thallium(I)- and thallium(III)-mediated rat pheochromocytoma (PC12) cell apoptosis.

    PubMed

    Pino, María Teresa Luján; Marotte, Clarisa; Verstraeten, Sandra Viviana

    2017-03-01

    We have reported recently that the proliferation of PC12 cells exposed to micromolar concentrations of Tl(I) or Tl(III) has different outcomes, depending on the absence (EGF(-) cells) or the presence (EGF(+) cells) of epidermal growth factor (EGF) added to the media. In the current work, we investigated whether EGF supplementation could also modulate the extent of Tl(I)- or Tl(III)-induced cell apoptosis. Tl(I) and Tl(III) (25-100 μM) decreased cell viability in EGF(-) but not in EGF(+) cells. In EGF(-) cells, Tl(I) decreased mitochondrial potential, enhanced H2O2 generation, and activated mitochondrial-dependent apoptosis. In addition, Tl(III) increased nitric oxide production and caused a misbalance between the anti- and pro-apoptotic members of Bcl-2 family. Tl(I) increased ERK1/2, JNK, p38, and p53 phosphorylation in EGF(-) cells. In these cells, Tl(III) did not affect ERK1/2 and JNK phosphorylation but increased p53 phosphorylation that was related to the promotion of cell senescence. In addition, this cation significantly activated p38 in both EGF(-) and EGF(+) cells. The specific inhibition of ERK1/2, JNK, p38, or p53 abolished Tl(I)-mediated EGF(-) cell apoptosis. Only when p38 activity was inhibited, Tl(III)-mediated apoptosis was prevented in EGF(-) and EGF(+) cells. Together, current results indicate that EGF partially prevents the noxious effects of Tl by preventing the sustained activation of MAPKs signaling cascade that lead cells to apoptosis and point to p38 as a key mediator of Tl(III)-induced PC12 cell apoptosis.

  14. The Aqueous Extract of Rhizome of Gastrodia elata Protected Drosophila and PC12 Cells against Beta-Amyloid-Induced Neurotoxicity

    PubMed Central

    Ng, Chun-Fai; Ko, Chun-Hay; Koon, Chi-Man; Xian, Jia-Wen; Leung, Ping-Chung; Fung, Kwok-Pui; Chan, Ho Yin Edwin; Lau, Clara Bik-San

    2013-01-01

    This study aims to investigate the neuroprotective effect of the rhizome of Gastrodia elata (GE) aqueous extract on beta-amyloid(Aβ)-induced toxicity in vivo and in vitro. Transgenic Drosophila mutants with Aβ-induced neurodegeneration in pan-neuron and ommatidia were used to determine the efficacy of GE. The antiapoptotic and antioxidative mechanisms of GE were also studied in Aβ-treated pheochromocytoma (PC12) cells. In vivo studies demonstrated that GE (5 mg/g Drosophila media)-treated Drosophila possessed a longer lifespan, better locomotor function, and less-degenerated ommatidia when compared with the Aβ-expressing control (all P < 0.05). In vitro studies illustrated that GE increased the cell viability of Aβ-treated PC12 cells in dose-dependent manner, probably through attenuation of Aβ-induced oxidative and apoptotic stress. GE also significantly upregulated the enzymatic activities of catalase, superoxide dismutase, and glutathione peroxidase, leading to the decrease of reactive oxidation species production and apoptotic marker caspase-3 activity. In conclusion, our current data presented the first evidence that the aqueous extract of GE was capable of reducing the Aβ-induced neurodegeneration in Drosophila, possibly through inhibition of apoptosis and reduction of oxidative stress. GE aqueous extract could be developed as a promising herbal agent for neuroprotection and novel adjuvant therapies for Alzheimer's disease. PMID:24174977

  15. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

    PubMed Central

    Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

    2012-01-01

    PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

  16. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+ homeostasis

    PubMed Central

    Liu, Yuan; Wang, Xue-chun; Hu, Dan; Huang, Shu-ran; Li, Qing-shu; Li, Zhi; Qu, Yan

    2016-01-01

    Heat shock protein 70 (HSP70) maintains Ca2+ homeostasis in PC12 cells, which may protect against apoptosis; however, the mechanisms of neuroprotection are unclear. Therefore, in this study, we examined Ca2+ levels in PC12 cells transfected with an exogenous lentiviral HSP70 gene expression construct, and we subsequently subjected the cells to ischemia-hypoxia/reoxygenation injury. HSP70 overexpression increased neuronal viability and ATPase activity, and it decreased cellular reactive oxygen species levels and intracellular Ca2+ concentration after hypoxia/reoxygenation. HSP70 overexpression enhanced the protein and mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), but it decreased the protein and mRNA levels of inositol 1,4,5-trisphosphate receptor (IP3R), thereby leading to decreased intracellular Ca2+ concentration after ischemia-hypoxia/reoxygenation. These results suggest that exogenous HSP70 protects against ischemia-hypoxia/reoxygenation injury, at least in part, by maintaining cellular Ca2+ homeostasis, by upregulating SERCA expression and by downregulating IP3R expression. PMID:27630698

  17. Protective effect of ferulic acid against 2,2'-azobis(2-amidinopropane) dihydrochloride-induced oxidative stress in PC12 cells.

    PubMed

    Shen, Y; Zhang, H; Wang, L; Qian, H; Qi, Y; Miao, X; Cheng, L; Qi, X

    2016-01-31

    Oxidative stress is closely related to the pathogenesis of neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. However, the underlying antioxidant mechanisms of ferulic acid (FA) aganist oxidantive stress are poorly understood. We evaluated the potential protective effects of FA against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced damage in PC12 cells. Our results indicated that pretreatment with FA prior to AAPH exposure significantly increased PC12 cell survival, and also increased catalase and superoxide dismutase activity. Furthermore, FA treatment reduced cellular lactate dehydrogenase release and malondialdehyde levels. It attenuated AAPH-induced apoptosis in PC12 cells, as determined by flow cytometric detection of annexin V. Reductions in mitochondrial membrane potential and accumulation of intracellular Ca2+ were also inhibited by FA treatment. These findings suggested that FA protected PC12 cells against AAPH-induced oxidative stress, and may be a neuroprotective agent.

  18. Effects of selenium and topiramate on cytosolic Ca(2+) influx and oxidative stress in neuronal PC12 cells.

    PubMed

    Demirci, Seden; Kutluhan, Süleyman; Naziroğlu, Mustafa; Uğuz, Abdülhadi Cihangir; Yürekli, Vedat Ali; Demirci, Kadir

    2013-01-01

    It has been widely suggested that selenium (Se) deficiency play an important role in the pathophysiology of epilepsy. It has been reported that Se provides protection against the neuronal damage in patients and animals with epilepsy by restoring the antioxidant defense mechanism. The neuroprotective effects of topiramate (TPM) have been reported in several studies but the putative mechanism of action remains elusive. We investigated effects of Se and TPM in neuronal PC12 cell by evaluating Ca(2+) mobilization, lipid peroxidation and antioxidant levels. PC12 cells were divided into eight groups namely control, TPM, Se, H(2)O(2), TPM + H(2)O(2), Se + H(2)O(2), Se + TPM and Se + TPM + H(2)O(2). The toxic doses and times of H(2)O(2), TPM and Se were determined by cell viability assay which is used to evaluate cell viability. Cells were incubated with 0.01 mM TPM for 5 h and 500 nM Se for 10 h. Then, the cells were exposed to 0.1 mM H(2)O(2) for 10 h before analysis. The cells in all groups except control, TPM and Se were exposed to H(2)O(2) for 15 min before analysis. Cytosolic Ca(2+) release and lipid peroxidation levels were higher in H(2)O(2) group than in control, Se and TPM combination groups although their levels were decreased by incubation of Se and TPM combination. However, there is no difference on Ca(2+) release in TPM group. Glutathione peroxidase activity, reduced glutathione and vitamin C levels in the cells were lower in H(2)O(2) group than in control, Se and TPM groups although their values were higher in the cells incubated with Se and TPM groups than in H(2)O(2) groups. In conclusion, these results indicate that Se induced protective effects on oxidative stress in PC12 cells by modulating cytosolic Ca(2+) influx and antioxidant levels. TPM modulated also lipid peroxidation and glutathione and vitamin C concentrations in the cell system.

  19. rAAV/ABAD-DP-6His attenuates oxidative stress-induced injury of PC12 cells

    PubMed Central

    Jia, Mingyue; Wang, Mingyu; Yang, Yi; Chen, Yixin; Liu, Dujuan; Wang, Xu; Song, Lei; Wu, Jiang; Yang, Yu

    2014-01-01

    Our previous studies have revealed that amyloid β (Aβ)-binding alcohol dehydrogenase (ABAD) decoy peptide antagonizes Aβ42-induced neurotoxicity. However, whether it improves oxidative stress injury remains unclear. In this study, a recombinant adenovirus constitutively secreting and expressing Aβ-ABAD decoy peptide (rAAV/ABAD-DP-6His) was successfully constructed. Our results showed that rAAV/ABAD-DP-6His increased superoxide dismutase activity in hydrogen peroxide-induced oxidative stress-mediated injury of PC12 cells. Moreover, rAAV/ABAD-DP-6His decreased malondialdehyde content, intracellular Ca2+ concentration, and the level of reactive oxygen species. rAAV/ABAD-DP-6His maintained the stability of the mitochondrial membrane potential. In addition, the ATP level remained constant, and apoptosis was reduced. Overall, the results indicate that rAAV/ABAD-DP-6His generates the fusion peptide, Aβ-ABAD decoy peptide, which effectively protects PC12 cells from oxidative stress injury induced by hydrogen peroxide, thus exerting neuroprotective effects. PMID:25206842

  20. Curcumin micelles improve mitochondrial function in neuronal PC12 cells and brains of NMRI mice - Impact on bioavailability.

    PubMed

    Hagl, Stephanie; Kocher, Alexa; Schiborr, Christina; Kolesova, Natalie; Frank, Jan; Eckert, Gunter P

    2015-10-01

    Curcumin, a polyphenolic compound abundant in the rhizome of Curcuma longa, has been reported to have various beneficial biological and pharmacological activities. Recent research revealed that curcumin might be valuable in the prevention and therapy of numerous disorders including neurodegenerative diseases like Alzheimer's disease. Due to its low absorption and quick elimination from the body, curcumin bioavailability is rather low which poses major problems for the use of curcumin as a therapeutic agent. There are several approaches to ameliorate curcumin bioavailability after oral administration, amongst them simultaneous administration with secondary plant compounds, micronization and micellation. We examined bioavailability in vivo in NMRI mice and the effects of native curcumin and a newly developed curcumin micelles formulation on mitochondrial function in vitro in PC12 cells and ex vivo in isolated mouse brain mitochondria. We found that curcumin micelles improved bioavailability of native curcumin around 10- to 40-fold in plasma and brain of mice. Incubation with native curcumin and curcumin micelles prevented isolated mouse brain mitochondria from swelling, indicating less mitochondrial permeability transition pore (mPTP) opening and prevention of injury. Curcumin micelles proved to be more efficient in preventing mitochondrial swelling in isolated mouse brain mitochondria and protecting PC12 cells from nitrosative stress than native curcumin. Due to their improved effectivity, curcumin micelles might be a suitable formulation for the prevention of mitochondrial dysfunction in brain aging and neurodegeneration.

  1. Carbon fiber nanoelectrodes applied to microchip electrophoresis amperometric detection of neurotransmitter dopamine in rat pheochromocytoma (PC12) cells.

    PubMed

    Cheng, Han; Huang, Wei-Hua; Chen, Rong-Sheng; Wang, Zong-Li; Cheng, Jie-Ke

    2007-05-01

    Microelectrodes have been adopted in electrochemical detection for CE or microchip CE in recent years. In this paper, the use of nanoelectrodes (with tip diameter of 100-300 nm) as the electrochemical detector in microchip CE is firstly reported. The experimental results indicated that both the sensitivity and resolution of microchip CE with the carbon fiber nanoelectrode (CFNE) amperometric detection have been improved markedly comparing with the traditional microelectrodes. The detection limit of dopamine (S/N = 3) is 5.9x10(-8) M, which is one or two orders of magnitude lower than that reported so far, and the resolution of dopamine (DA) and isoprenaline (IP) has also improved from 0.6 (using 7 mum carbon fiber microelectrodes, CFME) to 1.0. We assembled a novel and easily operated microchip CE system with end-column amperometric detection, which allows the convenient and fast replacement of the passivated electrodes. Under the optimized condition, the RSDs of peak height and migration time are 1.47 and 0.31%, respectively (n = 40), indicating that the system displays excellent reproducibility. The nanoelectrode-based microchip CE system has been successfully applied to the determination of DA in cultured rat pheochromocytoma (PC12) cells, and the average content of DA in an individual PC12 cell is 0.54 +/- 0.07 fmol, which is in good agreement with that reported in the literature.

  2. NGF-induction of the metalloproteinase-transin/stromelysin in PC12 cells: involvement of multiple protein kinases.

    PubMed

    Machida, C M; Scott, J D; Ciment, G

    1991-09-01

    In previous work, we found that nerve growth factor (NGF) induced expression of the mRNA transcript encoding the metalloproteinase transin/stromelysin in PC12 cells. Transin was found, moreover, to be a "late" gene product whose expression correlated with neurites extension. In this study, various aspects of the NGF intracellular signaling pathway in PC12 cells are investigated. We show that the protein kinase inhibitor staurosporine, but not various other kinase inhibitors, specifically blocked the NGF induction of transin. Preliminary characterization of this staurosporine-sensitive kinase suggest that it does not correspond to a tyrosine kinase, nor various serine kinases, and that it is involved both at the transcriptional and posttranscriptional levels of transin gene regulation. In contrast to these effects of staurosporine, various activators of protein kinases C and A augmented the NGF induction of transin. Similar effects of these kinase inhibitors and activators were also observed with the expression of various immediate-early genes that have been proposed to mediate the transcriptional effects of NGF, including c-fos and c-jun. These data suggest, therefore, that the NGF induction of transin mRNA expression involves multiple protein kinases acting at a number of postreceptor regulatory steps in the NGF signaling pathway.

  3. Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

    PubMed

    Makuch, Edyta; Kuropatwa, Marianna; Kurowska, Ewa; Ciekot, Jaroslaw; Klopotowska, Dagmara; Matuszyk, Janusz

    2014-07-05

    Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.

  4. Nerve growth factor-sensitive S6 kinase in cell-free extracts from PC12 cells

    SciTech Connect

    Matsuda, Y.; Nakanishi, N.; Dickens, G.; Guroff, G.

    1986-05-01

    Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a 2-10 fold increase in the ability to phosphorylate the ribosomal protein S6. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of (/sup 32/P) from (/sup 32/P)ATP into S6 occurred after 4-7 minutes of nerve growth factor treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000 and is not inhibited by the inhibitor of cAMP-dependent protein kinase, NaCl, or trifluoperazine, nor is it activated by the addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase, protein kinase C, nor the result of proteolytic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cyclic AMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. But the effect of NGF in increasing S6 kinase activity cannot be mimicked by treatment of control extract with cAMP-dependent protein kinase in vitro. Thus, it is unlikely to result from the phosphorylation of a less active form of the S6 kinase by a cAMP-dependent protein kinase.

  5. Efficient repairing effect of PEG based tri-block copolymer on mechanically damaged PC12 cells and isolated spinal cord.

    PubMed

    Rad, Iman; Mobasheri, Hamid; Najafi, Farhood; Rezaei, Maryam

    2014-06-01

    Membrane sealing effects of polymersomes made of tri-block copolymer, PEG-co-FA/SC-co-PEG, (PFSP) were studied on isolated spinal cord strips, PC12 cell lines and artificial bilayer following mechanical impact implemented by aneurism clip, sonication and electric shock, respectively. The homogeneity and size of PFSP, membrane permeability and cell viability were assessed by dynamic light scattering, LDH release and MTT assays. According to the results, the biocompatible, physico-chemical, size, surface charge and amphipathic nature of PFSP polymersome makes it an ideal macromolecule to rapidly reseal damaged membranes of cells in injured spinal cord as well as in culture medium. Compound action potentials recorded from intentionally damaged spinal cord strips incubated with PFSP showed restoration of neural excitability by 82.24 % and conduction velocity by 96.72 % after 5 min that monitored in real time. Thus, they triggered efficient instant and sustained sealing of membrane and reactivation of temporarily inactivated axons. Treatment of ultrasonically damaged PC12 cells by PFSP caused efficient cell membrane repair and led to their increased viability. The optimum effects of PFSP on stabilization and impermeabilizing of the lipid bilayer occurred at the same concentrations applied to the damaged cells and spinal cord fibers and was approved by restoration of membrane conductance and calcein release manifested by NanoDrop technique. The unique physico-chemical characteristics of novel polymersomes introduced here, make them capable to reorganize membrane lipid molecules, reseal the breaches and restore the hydrophobic insulation in spinal cord damaged cells. Thus, they might be considered in the clinical treatment of SCI at early stages.

  6. Salvianolic acid Y: a new protector of PC12 cells against hydrogen peroxide-induced injury from Salvia officinalis.

    PubMed

    Gong, Jun; Ju, Aichun; Zhou, Dazheng; Li, Dekun; Zhou, Wei; Geng, Wanli; Li, Bing; Li, Li; Liu, Yanjie; He, Ying; Song, Meizhen; Wang, Yunhua; Ye, Zhengliang; Lin, Ruichao

    2015-01-06

    Salvianolic acid Y (TSL 1), a new phenolic acid with the same planar structure as salvianolic acid B, was isolated from Salvia officinalis. The structural elucidation and stereochemistry determination were achieved by spectroscopic and chemical methods, including 1D, 2D-NMR (1H-1H COSY, HMQC and HMBC) and circular dichroism (CD) experiments. The biosynthesis pathway of salvianolic acid B and salvianolic acid Y (TSL 1) was proposed based on structural analysis. The protection of PC12 cells from injury induced by H2O2 was assessed in vitro using a cell viability assay. Salvianolic acid Y (TSL 1) protected cells from injury by 54.2%, which was significantly higher than salvianolic acid B (35.2%).

  7. Spontaneous proliferative lesions of the adrenal medulla in aging Long-Evans rats. Comparison to PC12 cells, small granule-containing cells, and human adrenal medullary hyperplasia.

    PubMed

    Tischler, A S; DeLellis, R A; Perlman, R L; Allen, J M; Costopoulos, D; Lee, Y C; Nunnemacher, G; Wolfe, H J; Bloom, S R

    1985-10-01

    Aging rats of the Long-Evans strain spontaneously develop diffuse and nodular hyperplasia of the adrenal medulla in association with other abnormalities commonly encountered in human multiple endocrine neoplasia syndromes. The cells which comprise the adrenal nodules resemble those in the parent tumor of the rat PC12 pheochromocytoma cell line in that they show varying degrees of spontaneous or nerve growth factor-induced neurite outgrowth in culture and they contain little or no epinephrine. In addition, cells from at least some of the nodules contain immunoreactive neurotensin and neuropeptide-Y, which are also found in PC12 cells. There are a number of striking resemblances between the cells in adrenal nodules and the small granule-containing cells in the normal rodent adrenal. The findings suggest that spontaneous rat adrenal medullary nodules and PC12 cells might be derived from small granule-containing cells, or that cells within the nodules might regain properties of immature chromaffin cells and acquire characteristics of small granule-containing cells and of PC12 cells in the course of neoplastic progression. They further suggest a possible relationship between proliferative capacity and neurotransmitter phenotype in the adult rat adrenal medulla. By virtue of their sparse epinephrine content and their small granules, the cells in adrenal medullary nodules of Long-Evans rats differ from those in adrenal medullary nodules of humans with multiple endocrine neoplasia syndromes.

  8. NGF induces the expression of group IIA secretory phospholipase A2 in PC12 cells: the newly synthesized enzyme is addressed to growing neurites.

    PubMed

    Nardicchi, Vincenza; Ferrini, Monica; Pilolli, Francesca; Angeli, Emanuela Biagioni; Persichetti, Emanuele; Beccari, Tommaso; Mannucci, Roberta; Arcuri, Cataldo; Donato, Rosario; Dorman, Robert V; Goracci, Gianfrancesco

    2014-08-01

    We proposed that group IIA secretory phospholipase A(2) (GIIA) participates in neuritogenesis based on our observations that the enzyme migrates to growth cones and neurite tips when PC12 cells are induced to differentiate by nerve growth factor (NGF) (Ferrini et al., Neurochem Res 35:2168-2174, 2010). The involvement of other secretory PLA(2) isoforms in neuronal development has been suggested by others but through different mechanisms. In the present study, we compared the subcellular distribution of GIIA and group X sPLA(2) (GX) after stimulation of PC12 cells with NGF. We found that GIIA, but not GX, localized at the neuritic tips after treatment with NGF, as demonstrated by immunofluorescence analysis. We also found that NGF stimulated the expression and the activity of GIIA. In addition, NGF induced the expressed myc-tagged GIIA protein to migrate to neurite tips in its active form. We propose that GIIA expression, activity, and subcellular localization is regulated by NGF and that the enzyme may participate in neuritogenesis through intracellular mechanisms, most likely by facilitating the remodelling of glycerophospholipid molecular species by deacylation-reacylation reactions necessary for the incorporation of polyunsaturated fatty acids.

  9. Neurotropin promotes NGF signaling through interaction of GM1 ganglioside with Trk neurotrophin receptor in PC12 cells.

    PubMed

    Fukuda, Yu; Fukui, Takao; Hikichi, Chika; Ishikawa, Tomomasa; Murate, Kenichiro; Adachi, Takeshi; Imai, Hideki; Fukuhara, Koki; Ueda, Akihiro; Kaplan, Allen P; Mutoh, Tatsuro

    2015-01-30

    Activation of the high-affinity nerve growth factor (NGF) receptor Trk occurs through multiple processes consisted of translocation and clustering within the plasma membrane lipid rafts, dimerization and autophosphorylation. Here we found that a nonprotein extract of inflamed rabbit skin inoculated with vaccinia virus (Neurotropin(®)) enhanced efficiency of NGF signaling. In rat pheochromocytoma PC12 cells overexpressing Trk (PCtrk cells), Neurotropin augmented insufficient neurite outgrowth observed at suboptimal concentration of NGF (2ng/mL) in a manner depending on Trk kinase activity. Cellular exposure to Neurotropin resulted in an accumulation of Trk-GM1 complexes without affecting dimerization or phosphorylation states of Trk. Following NGF stimulation, Neurotropin significantly facilitated the time course of NGF-induced Trk autophosphorylation. These observations provide a unique mechanism controlling efficiency of NGF signaling, and raise the therapeutic potential of Neurotropin for various neurological conditions associated with neurotrophin dysfunction.

  10. The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

    SciTech Connect

    Sato, Mai; Kitaguchi, Tetsuya; Ikematsu, Kazuya; Kakeyama, Masaki; Murata, Masayuki; Sato, Ken; Tsuboi, Takashi

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer Regulation of exocytosis by Rho GTPase Cdc42. Black-Right-Pointing-Pointer Cdc42 increases the number of fusion events from newly recruited vesicles. Black-Right-Pointing-Pointer Cdc42 increases the number of exocytosis-competent dense-core vesicles. -- Abstract: Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.

  11. Hexabromocyclododecane inhibits depolarization-induced increase in intracellular calcium levels and neurotransmitter release in PC12 cells.

    PubMed

    Dingemans, Milou M L; Heusinkveld, Harm J; de Groot, Aart; Bergman, Ake; van den Berg, Martin; Westerink, Remco H S

    2009-02-01

    Environmental levels of the brominated flame retardant (BFR) hexabromocyclododecane (HBCD) have been increasing. HBCD has been shown to cause adverse effects on learning and behavior in mice, as well as on dopamine uptake in rat synaptosomes and synaptic vesicles. For other BFRs, alterations in the intracellular Ca(2+) homeostasis have been observed. Therefore, the aim of this study was to investigate whether the technical HBCD mixture and individual stereoisomers affect the intracellular Ca(2+) concentration ([Ca(2+)](i)) in a neuroendocrine in vitro model (PC12 cells). [Ca(2+)](i) and vesicular catecholamine release were measured using respectively single-cell Fura-2 imaging and amperometry. Exposure of PC12 cells to the technical HBCD mixture or individual stereoisomers did neither affect basal [Ca(2+)](i), nor the frequency of basal neurotransmitter release. However, exposure to HBCD (0-20 microM) did cause a dose-dependent reduction of a subsequent depolarization-evoked increase in [Ca(2+)](i). This effect was apparent only when HBCD was applied at least 5 min before depolarization (maximum effect after 20 min exposure). The effects of alpha- and beta-HBCD were comparable to that of the technical mixture, whereas the inhibitory effect of gamma-HBCD was larger. Using specific blockers of L-, N- or P/Q-type voltage-gated Ca(2+) channels (VGCCs) it was shown that the inhibitory effect of HBCD is not VGCC-specific. Additionally, the number of cells showing depolarization-evoked neurotransmitter release was markedly reduced following HBCD exposure. Summarizing, HBCD inhibits depolarization-evoked [Ca(2+)](i) and neurotransmitter release. As increasing HBCD levels should be anticipated, these findings justify additional efforts to establish an adequate exposure, hazard and risk assessment.

  12. Actin and dynamin recruitment and the lack thereof at exo- and endocytotic sites in PC12 cells.

    PubMed

    Felmy, Felix

    2009-06-01

    Protein recruitment during endocytosis is well characterized in fibroblasts. Since fibroblasts do not engage in regulated exocytosis, only information about protein recruitment during constitutive endocytosis is provided. Furthermore, the cortical actin of fibroblasts is characterized by stress fibers rather than a thick cortical meshwork. A cell model, which differs in these features, could provide insight into the heterogeneity of protein recruitment to constitutive and exocytosis coupled endocytotic areas. Therefore, this study investigates the sequence of protein recruitment in PC12 cells, a well documented exocytotic cell model with thick actin cortex. Using real time total-internal-reflection fluorescence microscopy it was found that at the plasma membrane steady, but not transient, dynamin-1-EGFP or -mCherry fluorescence spots that rapidly dimmed coincided with markers for constitutive endocytotic such as clathrin-LC-dsRed and transferrin-receptor-pHluorin. Clathrin-LC-dsRed and dynamin-1-EGFP were further used to determine the temporal sequence of protein recruitment to areas of constitutive endocytosis. mCherry- and EGFP-beta-actin, Arp-3-EGFP and EGFP-mAbp1 were slowly recruited before the dynamin-1-mCherry fluorescence dimmed, but their fluorescence peaked after the loss of clathrin-LC-dsRed commenced. Furthermore, mCherry-beta-actin fluorescence increased before exocytosis, indicating redistribution prior to release. Also, no average dynamin-1-mCherry recruitment was observed within 50 s to regions of exocytosis marked by NPY-mGFP. This indicates that the temporal-spatial coupling between regulated exo-and endocytosis is rather limited in PC12 cells. Furthermore, the time course of the protein recruitment to constitutive endocytotic sites might depend on the subcellular morphology such as the size of the actin cortex.

  13. Extracellular α-synuclein leads to microtubule destabilization via GSK-3β-dependent Tau phosphorylation in PC12 cells.

    PubMed

    Gąssowska, Magdalena; Czapski, Grzegorz A; Pająk, Beata; Cieślik, Magdalena; Lenkiewicz, Anna M; Adamczyk, Agata

    2014-01-01

    α-Synuclein (ASN) plays an important role in pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. Novel and most interesting data showed elevated tauopathy in PD and suggested relationship between ASN and Tau protein. However, the mechanism of ASN-evoked Tau protein modification is not fully elucidated. In this study we investigated the role of extracellular ASN in Tau hyperphosphorylation in rat pheochromocytoma (PC12) cells and the involvement of glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase 5 (CDK5) in ASN-dependent Tau modification. Our results indicated that exogenously added ASN increases Tau phosphorylation at Ser396. Accordingly, the GSK-3β inhibitor (SB-216763) prevented ASN-evoked Tau hyperphosphorylation, but the CDK5 inhibitor had no effect. Moreover, western blot analysis showed that ASN affected GSK-3β via increasing of protein level and activation of this enzyme. GSK-3β activity evaluated by its phosphorylation status assay showed that ASN significantly increased the phosphorylation of this enzyme at Tyr216 with parallel decrease in phosphorylation at Ser9, indicative of stimulation of GSK-3β activity. Moreover, the effect of ASN on microtubule (MT) destabilization and cell death with simultaneous the involvement of GSK-3β in these processes were analyzed. ASN treatment increased the amount of free tubulin and concomitantly reduced the amount of polymerized tubulin and SB-216763 suppressed these ASN-induced changes in tubulin, indicating that GSK-3β is involved in ASN-evoked MT destabilization. ASN-induced apoptotic processes lead to decrease in PC12 cells viability and SB-216763 protected those cells against ASN-evoked cytotoxicity. Concluding, extracellular ASN is involved in GSK-3β-dependent Tau hyperphosphorylation, which leads to microtubule destabilization. GSK-3β inhibition may be an effective strategy for protecting against ASN-induced cytotoxicity.

  14. Interaction of new antidepressants with sigma-1 receptor chaperones and their potentiation of neurite outgrowth in PC12 cells.

    PubMed

    Ishima, Tamaki; Fujita, Yuko; Hashimoto, Kenji

    2014-03-15

    The sigma-1 receptor chaperone located in the endoplasmic reticulum (ER) may be implicated in the mechanistic action of some antidepressants. The present study was undertaken to examine whether new antidepressant drugs interact with the sigma-1 receptor chaperone. First, we examined the effects of selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, citalopram and escitalopram), serotonin and noradrenaline reuptake inhibitors (SNRIs) (duloxetine, venlafaxine, milnacipran), and mirtazapine, a noradrenaline and specific serotonergic antidepressant (NaSSA), on [(3)H](+)-pentazocine binding to rat brain membranes. Then, we examined the effects of these drugs on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The order of potency for drugs at the sigma-1 receptor chaperone was as follows: fluvoxamine>sertraline>fluoxetine>escitalopram>citalopram>paroxetine>duoxetine. Venlafaxine, milnacipran, and mirtazapine showed very weak affinity for this chaperone. Furthermore, fluvoxamine, fluoxetine, escitalopram, and mirtazapine significantly potentiated NGF-induced neurite outgrowth in cell assays, and the effects of all these drugs, excluding mirtazapine, were antagonized by NE-100, a selective antagonist of the sigma-1 receptor chaperone. Moreover, the effects of fluvoxamine and fluoxetine on neurite outgrowth were also antagonized by sertraline, indicating that sertraline may be an antagonist at the sigma-1 receptor chaperone. The effect of mirtazapine on neurite outgrowth was antagonized by the selective 5-hydroxytryptamine1A receptor antagonist WAY-100635. These findings suggest that activation at the sigma-1 receptor chaperone may be involved in the action of some SSRIs, such as fluvoxamine, fluoxetine and escitalopram. In contrast, mirtazapine independently potentiated neurite outgrowth in PC12 cells, indicating that this beneficial effect may mediate its pharmacological effect.

  15. Nitric oxide and histone deacetylases modulate cocaine-induced mu-opioid receptor levels in PC12 cells

    PubMed Central

    2012-01-01

    Background Cocaine exposure has been reported to alter central μ-opioid receptor (MOR) expression in vivo. The present study employed an in vitro cellular model to explore possible mechanisms that may be involved in this action of cocaine. Methods To assess the effects of cocaine on MOR levels, two treatment regimens were tested in PC12 cells: single continuous or multiple intermittent. MOR protein levels were assessed by western blot analysis and quantitative PCR was used to determine relative MOR mRNA expression levels. To evaluate the role of nitric oxide (NO) and histone acetylation in cocaine-induced MOR expression, cells were pre-treated with the NO synthase inhibitor Nω-nitro-L-arginine methylester (L-NAME) or the non-selective histone acetyltransferase inhibitor curcumin. Results Both cocaine treatment regimens significantly increased MOR protein levels and protein stability, but only multiple intermittent treatments increased MOR mRNA levels as well as c-fos mRNA levels and activator protein 1 binding activity. Both regimens increased NO production, and pre-treatment with L-NAME prevented cocaine-induced increases in MOR protein and mRNA levels. Single and multiple cocaine treatment regimens inhibited histone deacetylase activity, and pre-treatment with curcumin prevented cocaine-induced up-regulation of MOR protein expression. Conclusions In the PC12 cell model, both NO and histone deacetylase activity regulate cocaine-induced MOR expression at both the transcriptional and post-transcriptional levels. Based on these novel findings, it is hypothesized that epigenetic mechanisms are implicated in cocaine’s action on MOR expression in neurons. PMID:23079001

  16. The synthetic cannabinoid HU-210 attenuates neural damage in diabetic mice and hyperglycemic pheochromocytoma PC12 cells.

    PubMed

    Dagon, Yossi; Avraham, Yosefa; Link, Gabriela; Zolotarev, Olga; Mechoulam, Raphael; Berry, Elliot M

    2007-08-01

    Diabetic neuropathy (DN) is a common complication of diabetes mellitus resulting in cognitive dysfunction and synaptic plasticity impairment. Hyperglycemia plays a critical role in the development and progression of DN, through a number of mechanisms including increased oxidative stress. Cannabinoids are a diverse family of compounds which can act as antioxidative agents and exhibit neuroprotective properties. We investigated the effect of the synthetic cannabinoid HU-210 on brain function of streptozotocin (STZ)-induced diabetic mice. These animals exhibit hyperglycemia, increased cerebral oxidative stress and impaired brain function. HU-210, through a receptor independent pathway, alleviates the oxidative damage and cognitive impairment without affecting glycemic control. To study the neuroprotective mechanism(s) involved, we cultured PC12 cells under hyperglycemic conditions. Hyperglycemia enhanced oxidative stress and cellular injuries were all counteracted by HU-210-in a dose dependent manner. These results suggest cannabinoids might have a therapeutic role in the management of the neurological complications of diabetes.

  17. Protective effects of fractions from Artemisia biennis hydro-ethanolic extract against doxorubicin-induced oxidative stress and apoptosis in PC12 cells

    PubMed Central

    Mojarrab, Mahdi; Mehrabi, Mehran; Ahmadi, Farahnaz; Hosseinzadeh, Leila

    2016-01-01

    Objective(s): This study was designed to indicate whether different fractions from Artemisia biennis hydroethanolic extract could provide cytoprotection against oxidative stress and apoptosis induced by doxorubicin (DOX) in rat pheochromocytoma cell line (PC12). Material and Methods: Cell viability was determined by MTT assay. Also, activation of caspase-3 and superoxide dismutase were evaluated by spectrophotometry. Detection of reactive oxygen species (ROS) and measurement of mitochondrial membrane potential (MMP) were performed by flowcytometry. Results: Treatment of PC12 cells with DOX reduced viability dose dependently. For evaluation of the effect of fractions (A-G) on DOX-induced cytotoxicity, PC12 cells were pretreated for 24 hr with the A. biennis fractions and then cells were treated with DOX. The fractions C and D increased PC12 cells viability significantly compared to DOX treated cells. Moreover, pretreatment with fractions C and D for 24 hr attenuated DOX-mediated apoptosis and the anti-apoptotic action of A. biennis fractions was partially dependent on inhibition of caspase 3 activity and also increasing the mitochondrial membrane potential (MMP). Selected A. biennis fractions also suppressed the generation of ROS and increased superoxide dismutase (SOD) activity. Conclusion: Taken together our observation indicated that subtoxic concentration of aforementioned fractions of A. biennis hydroetanolic extract has protective effect against apoptosis induced by DOX in PC12 cell. The results highlighted that fractions C and D may exert cytoprotective effects through their antioxidant actions. PMID:27403257

  18. Pycnogenol Protects Against Rotenone-Induced Neurotoxicity in PC12 Cells Through Regulating NF-κB-iNOS Signaling Pathway.

    PubMed

    Gao, Bo; Chang, Chongwang; Zhou, Jie; Zhao, Tianzhi; Wang, Chao; Li, Chen; Gao, Guodong

    2015-10-01

    Parkinson's disease (PD) is a common neurodegenerative disorder characterized by dopaminergic neurons degeneration and oxidative damage may underlie this process. However, there are still no efficient drugs to cure the disease. Pycnogenol (PYC) isolated from the procyanidin-rich French maritime pine (Pinus maritime) bark has shown various antioxidant activities in previous studies. In this study, we explored its effect against rotenone (Rot)-induced neurotoxicity and the underlying mechanisms in PC12 cells. Using Rot-induced cell model of PD, we found that PYC treatment significantly increased cell viability and decreased cell apoptosis in Rot-treated PC12 cells in a dose-dependent manner. Furthermore, data showed that PYC markedly reduced inducible nitric oxide synthase (iNOS)-nitric oxide (NO) signaling in Rot-treated PC12 cells. Pretreatment with the iNOS-specific inhibitor significantly attenuated Rot-induced neurotoxicity. Moreover, PYC was found to be capable of reducing Rot-induced NF-κB activation. Blocking NF-κB signaling with its inhibitor mimicked the biological effect of PYC on Rot-induced iNOS and NO expression levels, as well as neurotoxicity in PC12 cells, suggesting that the NF-κB-iNOS signaling pathway was likely to participate in the PYC-mediated protective progress. Our results suggest that PYC protects against Rot-induced neurotoxicity in PC12 cells, and the mechanism may be associated with the downregulation of NF-κB-iNOS signaling pathway.

  19. Translationally Controlled Tumor Protein Stimulates Dopamine Release from PC12 Cells via Ca(2+)-Independent Phospholipase A₂ Pathways.

    PubMed

    Seo, Jihui; Maeng, Jeehye; Kim, Hwa-Jung

    2016-10-24

    The translationally controlled tumor protein (TCTP), initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF). TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12) cells. Treatment with recombinant TCTP (rTCTP) enhanced both basal and depolarization (50 mM KCl)-evoked [³H]dopamine release in concentration- and time-dependent manners. Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca(2+)]i), the rTCTP-driven effect on dopamine release was mediated by a Ca(2+)-independent pathway, as evidenced by the fact that Ca(2+)-modulating agents such as Ca(2+) chelators and a voltage-gated L-type Ca(2+)-channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A₂ (PLA₂) in rTCTP-induced dopamine release, the inhibitor for Ca(2+)-independent PLA₂ (iPLA₂) produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca(2+)-dependent cytosolic PLA₂ (cPLA₂) and secretory PLA₂ (sPLA₂) inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca(2+)-independent mechanism that involved PLA₂ in the process, suggesting the regulatory role of TCTP in the neuronal functions.

  20. Translationally Controlled Tumor Protein Stimulates Dopamine Release from PC12 Cells via Ca2+-Independent Phospholipase A2 Pathways

    PubMed Central

    Seo, Jihui; Maeng, Jeehye; Kim, Hwa-Jung

    2016-01-01

    The translationally controlled tumor protein (TCTP), initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF). TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12) cells. Treatment with recombinant TCTP (rTCTP) enhanced both basal and depolarization (50 mM KCl)-evoked [3H]dopamine release in concentration- and time-dependent manners. Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca2+]i), the rTCTP-driven effect on dopamine release was mediated by a Ca2+-independent pathway, as evidenced by the fact that Ca2+-modulating agents such as Ca2+ chelators and a voltage-gated L-type Ca2+-channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A2 (PLA2) in rTCTP-induced dopamine release, the inhibitor for Ca2+-independent PLA2 (iPLA2) produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca2+-dependent cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2) inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca2+-independent mechanism that involved PLA2 in the process, suggesting the regulatory role of TCTP in the neuronal functions. PMID:27783042

  1. EFFECT OF METHYL MERCURY CHLORIDE EXPOSURE ON PC12 CELL INTEGRIN EXPRESSION AND FUNCTION.

    EPA Science Inventory

    Integrins are heterodimeric transmembrane cell adhesion proteins composed of a and b protein subunits. They are important during brain development in a number of critical functions, including cell migration (Georges-Labouesse, et al., 1998), axonal elongation (Murase and Hayashi...

  2. Modulation of PC12 cell viability by forskolin-induced cyclic AMP levels through ERK and JNK pathways: an implication for L-DOPA-induced cytotoxicity in nigrostriatal dopamine neurons.

    PubMed

    Park, Keun Hong; Park, Hyun Jin; Shin, Keon Sung; Choi, Hyun Sook; Kai, Masaaki; Lee, Myung Koo

    2012-07-01

    The intracellular levels of cyclic AMP (cAMP) increase in response to cytotoxic concentrations of L-DOPA in PC12 cells, and forskolin that induces intracellular cAMP levels either protects PC12 cells from L-DOPA-induced cytotoxicity or enhances cytotoxicity in a concentration-dependent manner. This study investigated the effects of cAMP induced by forskolin on cell viability of PC12 cells, relevant to L-DOPA-induced cytotoxicity in Parkinson's disease therapy. The low levels of forskolin (0.01 and 0.1 μM)-induced cAMP increased dopamine biosynthesis and tyrosine hydroxylase (TH) phosphorylation, and induced transient phosphorylation of ERK1/2 within 1 h. However, at the high levels of forskolin (1.0 and 10 μM)-induced cAMP, dopamine biosynthesis and TH phosphorylation did not increase, but rapid differentiation in neurite-like formation was observed with a steady state. The high levels of forskolin-induced cAMP also induced sustained increase in ERK1/2 phosphorylation within 0.25-6 h and then led to apoptosis, which was apparently mediated by JNK1/2 and caspase-3 activation. Multiple treatment of PC12 cells with nontoxic L-DOPA (20 μM) for 4-6 days induced neurite-like formation and decreased intracellular dopamine levels by reducing TH phosphorylation. These results suggest that the low levels of forskolin-induced cAMP increased dopamine biosynthesis in cell survival via transient ERK1/2 phosphorylation. In contrast, the high levels of forskolin-induced cAMP induced differentiation via sustained ERK1/2 phosphorylation and then led to apoptosis. Taken together, the intracellular levels of cAMP play a dual role in cell survival and death through the ERK1/2 and JNK1/2 pathways in PC12 cells.

  3. Oncogene N-ras mediates selective inhibition of c-fos induction by nerve growth factor and basic fibroblast growth factor in a PC12 cell line.

    PubMed Central

    Thomson, T M; Green, S H; Trotta, R J; Burstein, D E; Pellicer, A

    1990-01-01

    A cell line was generated from U7 cells (a subline of PC12 rat pheochromocytoma cells) that contains a stably integrated transforming mouse N-ras (Lys-61) gene under the control of the long terminal repeat from mouse mammary tumor virus. Such cells, designated UR61, undergo neuronal differentiation upon exposure to nanomolar concentrations of dexamethasone, as a consequence of expression of the activated N-ras gene (I. Guerrero, A. Pellicer, and D.E. Burstein, Biochem, Biophys. Res. Commun. 150:1185-1192, 1988). Exposure of UR61 cells to either nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) results in a marked induction of c-fos RNA, with kinetics paralleling those of NGF- or bFGF-induced expression of c-fos RNA in PC12 cells. Dexamethasone-induced expression of activated N-ras p21 results in blocking of c-fos RNA induction by NGF or bFGF in a time-dependent manner. Activated N-ras p21-mediated inhibition of c-fos RNA induction in UR61 cells is selective for NGF and bFGF and is not due to selective degradation of c-fos RNA. Normal and transforming N-ras can trans activate the chloramphenicol acetyltransferase gene linked to mouse c-fos regulatory sequences when transient expression assays are performed. Our observations suggest that N-ras p21 selectively interacts with pathways involved in induction of c-fos expression which initiate at the receptors for NGF and bFGF. Images PMID:2108319

  4. The Neuroprotective Effects of Decursin Isolated from Angelica gigas Nakai Against Amyloid β-Protein-Induced Apoptosis in PC 12 Cells via a Mitochondria-Related Caspase Pathway.

    PubMed

    Li, Li; Du, Jikun; Zou, Liyi; Xia, Haishan; Wu, Tie; Kim, Yongho; Lee, Yongwoo

    2015-08-01

    Decursin, purified from Angelica gigas Nakai, has been proven to exert neuroprotective property. Previous study revealed decursin protected the PC12 cells from Aβ25-35-induced oxidative cytotoxicity. The present study aimed to investigate whether decursin could protect PC12 cells from apoptosis caused by Aβ. Our results indicated that pretreatment of PC12 cells with decursin significantly inhibited Aβ25-35-induced cytotoxicity and apoptosis. The mechanism of action is likely to reverse Aβ25-35-induced mitochondrial dysfunction, including the reduction of mitochondrial membrane potential, the inhibition of reactive oxygen species production, and the decrease of mitochondrial release of cytochrome c in PC12 cells. In addition, decursin significantly suppressed the activity of caspase-3 and moderated the ratio of Bcl-2/Bax induced by Aβ25-35. These findings indicate that decursin exerts a neuroprotective effect against Aβ25-35-induced neurotoxicity in PC12 cells, at least in part, via suppressing the mitochondrial pathway of cellular apoptosis.

  5. Effect of Zirconium Dioxide Nanoparticles on Glutathione Peroxidase Enzyme in PC12 and N2a Cell Lines

    PubMed Central

    Asadpour, Elham; Sadeghnia, Hamid Reza; Ghorbani, Ahmad; Boroushaki, Mohammad Taher

    2014-01-01

    Today, special attention is paid to the use of zirconium dioxide nanoparticle (nano-ZrO2), a neutral bioceramic metal, particularly for drug and gene delivery in medicine. However, there are some reports implying that use of nano-ZrO2 is associated with cytotoxic effects like inhibiting the cell proliferation, DNA damage and apoptosis. In the present study, we examined whether nano-ZrO2 alters cell viability and glutathione peroxidase (GPx) activity in two neuronal cell lines. The PC12 and N2a cells were cultured in the absence or presence of varying concentrations (31.25-2000 µg/mL) of nano-ZrO2 for 12, 24 or 48 h. The cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and GPx activity was determined by quantifying the rate of oxidation of the reduced glutathione to the oxidized glutathione. Nano-ZrO2 caused a significant reduction in cell viability and GPx activity after 12, 24 and 48 h, as compared with control group. These effects were concentration dependent and started from 250 µg/mL. The present study demonstrated that nano-ZrO2, at concentrations of > 250 µg/mL, has antiproliferative effects via reducing the cell defense mechanism against oxidative stress. PMID:25587301

  6. Cyanidin suppresses amyloid beta-induced neurotoxicity by inhibiting reactive oxygen species-mediated DNA damage and apoptosis in PC12 cells

    PubMed Central

    Wang, Yi; Fu, Xiao-ting; Li, Da-wei; Wang, Kun; Wang, Xin-zhi; Li, Yuan; Sun, Bao-liang; Yang, Xiao-yi; Zheng, Zun-cheng; Cho, Nam Chun

    2016-01-01

    Amyloid beta (Aβ)-induced oxidative stress is a major pathologic hallmark of Alzheimer's disease. Cyanidin, a natural flavonoid compound, is neuroprotective against oxidative damage-mediated degeneration. However, its molecular mechanism remains unclear. Here, we investigated the effects of cyanidin pretreatment against Aβ-induced neurotoxicity in PC12 cells, and explored the underlying mechanisms. Cyanidin pretreatment significantly attenuated Aβ-induced cell mortality and morphological changes in PC12 cells. Mechanistically, cyanidin effectively blocked apoptosis induced by Aβ, by restoring the mitochondrial membrane potential via upregulation of Bcl-2 protein expression. Moreover, cyanidin markedly protected PC12 cells from Aβ-induced DNA damage by blocking reactive oxide species and superoxide accumulation. These results provide evidence that cyanidin suppresses Aβ-induced cytotoxicity, by preventing oxidative damage mediated by reactive oxide species, which in turn inhibits mitochondrial apoptosis. Our study demonstrates the therapeutic potential of cyanidin in the prevention of oxidative stress-mediated Aβ neurotoxicity. PMID:27335564

  7. Enantioselective Cytotoxicity Profile of o,p’-DDT in PC 12 Cells

    PubMed Central

    Zhang, Chunlong; Wen, Yuezhong; Liu, Weiping

    2012-01-01

    Background The continued uses of dichlordiphenyltrichloroethane (DDT) for indoor vector control in some developing countries have recently fueled intensive debates toward the global ban of this persistent legacy contaminant. Current approaches for ecological and health risk assessment has ignored the chiral nature of DDT. In this study by employing an array of cytotoxicity related endpoints, we investigated the enantioselective cytotoxicity of o,p’-DDT. Principal Findings we demonstrated for the first time that R-(−)-o,p’-DDT caused more neuron cell death by inducing more severe oxidative stress, which selectively imbalanced the transcription of stress-related genes (SOD1, SOD2, HSP70) and enzyme (superoxide dismutase and lactate dehydrogenase) activities, and greater cellular apoptosis compared to its enantiomer S-(+)-o,p’-DDT at the level comparable to malaria area exposure (parts per million). We further elucidated enantioselective modes of action using microarray combined with enzyme-linked immunosorbent assay. The enantioselective apoptosis might involve three signaling pathways via caspase 3, tumor protein 53 (p53) and NFkB. Conclusions Based on DDT stereochemistry and results reported for other chiral pesticides, our results pointed to the same directional enantioselectivity of chiral DDT toward mammalian cells. We proposed that risk assessment on DDT should consider the enantiomer ratio and enantioselectivities. PMID:22937105

  8. Identification of the alternative spliced form of the alpha 2/delta subunit of voltage sensitive Ca2+ channels expressed in PC12 cells.

    PubMed

    Gilad, B; Shenkar, N; Halevi, S; Trus, M; Atlas, D

    1995-07-07

    The alpha 2/delta subunit of voltage sensitive Ca2+ channels expressed in PC12 has been cloned and partially sequenced. The message observed in Northern blot analysis displays a 7.5 kb transcript, identical in size to mRNA of rabbit skeletal muscle and rat brain. The nucleotide sequence of the cloned alpha 2 subunit of the PC12 specific cDNA is > 99% identical to rat brain sequence and 85% to skeletal muscle. Reverse-transcriptase-polymerase chain reaction (RT-PCR) of the alternative splicing region identifies two deleted regions of 57 bp and 21 bp in PC12 expressed alpha 2/delta transcript. The alternative variant alpha 2e of alpha 2/delta subunit which is expressed in PC12 cells was previously identified in human embryonic kidney (HEK293) cells. RT-PCR analysis show two different sized alternative PCR fragments in rat lung and none in rat spleen, kidney and intestine. Antibodies prepared against a 19 amino acid peptide within the alternative spliced region effectively inhibits [3H]dopamine release in PC12 cells. This implies that the alternatively spliced region is positioned extracellularly and is involved in regulation of the L-type Ca2+ channel-mediated transmitter release.

  9. Involvement of caspase-3 and p38 mitogen-activated protein kinase in cobalt chloride-induced apoptosis in PC12 cells.

    PubMed

    Zou, Weiguo; Zeng, Jiping; Zhuo, Ming; Xu, Weijing; Sun, Lanying; Wang, Jinxing; Liu, Xinyuan

    2002-03-15

    Our previous study showed that cobalt chloride (CoCl2) could induce PC12 cell apoptosis and that the CoCl2-treated PC12 cells may serve as a simple in vitro model for the study of the mechanism of hypoxia-linked neuronal disorders. The aim of this study is to elucidate the mechanism of CoCl2-induced apoptosis in PC12 cells. Caspases are known to be involved in the apoptosis induced by various stimuli in many cell types. To investigate the involvement of caspases in CoCl2-induced apoptosis in PC12 cells, we generated PC12 cells that stably express the viral caspases inhibitor gene p35 and analyzed the effect of p35 on the process of apoptosis induced by CoCl2. We also examined the effect of cell-permeable peptide inhibitors of caspases. The results showed that the baculovirus p35 gene and the general caspases inhibitor Z-VAD-FMK significantly block apoptosis induced by CoCl2, confirming that caspase is involved in CoCl2-induced apoptosis. Further investigation showed that in this process the caspase-3-like activity is increased, as indicated by the cells' ability to cleave the fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-7-AMC and to degrade the DNA-repairing enzyme poly-(ADP-ribose) polymerase (PARP), an endogenous caspase-3 substrate. At the same time, caspase-3-specific inhibitors, namely, the peptide Ac-DEVD-CHO, Ac-DEVD-FMK, partially inhibit CoCl2-induced apoptosis. These findings suggested that caspase-3 or caspase-3-like proteases are involved in the apoptosis induced by CoCl2 in PC12 cells. Additionally, we have observed that another apoptotic marker, p38 mitogen-activated protein kinase (MAPK), is significantly activated in this process in a time-dependent manner and that a selective p38 MAPK inhibitor, SB203580, partially inhibits this cell death. The addition of SB203580 also partially suppresses caspase-3-like activity. All these results confirm that the CoCl2-treated PC12 cell is a useful in vitro model with which to study hypoxia-linked neuronal

  10. Protective effects of SKF-96365, a non-specific inhibitor of SOCE, against MPP+-induced cytotoxicity in PC12 cells: potential role of Homer1.

    PubMed

    Chen, Tao; Zhu, Jie; Zhang, Chi; Huo, Kai; Fei, Zhou; Jiang, Xiao-fan

    2013-01-01

    Parkinson's disease (PD) is the most common neurodegenerative movement disorder, characterized by loss of dopominergic (DA) neurons in substantia nigra pars compacta (SNpc), and can be experimentally mimicked by the neurotoxin MPP(+) in vitro models. In this study, we investigated the potential protective effect of SKF-96365, a non-specific inhibitor of SOCE (store-operated calcium entry), on MPP(+) induced cytotoxicity in PC12 cells. We found that pretreatment with SKF-96365 (10 µM and 50 µM) 30 min before injury significantly increased cell viability, decreased LDH release, prevented nuclear damage, and inhibited apoptotic cell death in MPP(+) stressed PC12 cells. The results of calcium image using the ratiometric calcium indicator Fura-2-AM also showed that SKF-96365 reduced the intracellular calcium overload induced by MPP(+) in PC12 cells. In addition, SKF-96365 decreased the expression of Homer1, a more recently discovered postsynaptic scaffolding protein with calcium modulating function, following MPP(+) administration in PC12 cells, while had no statistically significant effects on endoplasmic reticulum (ER) calcium concentration. Furthermore, overexpression of Homer1 by using recombinant lentivirus partly reversed protective effects of SKF-96365 against MPP(+) injury. The ER Ca(2+) release was further amplified and ER calcium recovery was delayed by Homer1 upregulation in PC12 cells following MPP(+) insult. Taken together, these data suggest that SKF-96365 protects PC12 cells against MPP(+) induced cytotoxicity, and this protection may be at least in part on the inhibition of intracellular calcium overload and suppression of Homer1-mediated ER Ca(2+) release.

  11. Minimal requirements for exocytosis. A study using PC 12 cells permeabilized with staphylococcal alpha-toxin

    SciTech Connect

    Ahnert-Hilger, G.; Bhakdi, S.; Gratzl, M.

    1985-10-15

    The membrane-permeabilizing effects of streptolysin O, staphylococcal alpha-toxin, and digitonin on cultured rat pheochromocytoma cells were studied. All three agents perturbed the plasma membrane, causing release of intracellular YWRb and uptake of trypan blue. In addition, streptolysin O and digitonin also damaged the membranes of secretory vesicles, including a parallel release of dopamine. In contrast, the effects of alpha-toxin appeared to be strictly confined to the plasma membrane, and no dopamine release was observed with this agent. The exocytotic machinery, however, remained intact and could be triggered by subsequent introduction of micromolar concentrations of Ca2+ into the medium. Dopamine release was entirely Ca2+ specific and occurred independent of the presence or absence of other cations or anions including K+ glutamate, K+ acetate, or Na+ chloride. Ca2+-induced exocytosis did not require the presence of Mg2+-ATP in the medium. The process was insensitive to pH alterations in the range pH 6.6-7.2, and appeared optimal at an osmolarity of 300 mosm/kg. Toxin permeabilization seems to be an excellent method for studying the minimal requirements for exocytosis.

  12. Comparative Study on the Protective Effects of Salidroside and Hypoxic Preconditioning for Attenuating Anoxia-Induced Apoptosis in Pheochromocytoma (PC12) Cells

    PubMed Central

    Hu, Yao; Lv, Xiumei; Zhang, Jing; Meng, Xianli

    2016-01-01

    Background Hypoxia is an important sign that can result from body injuries or a special condition such as being at a high altitude or deep water diving. In the current studies, hypoxic preconditioning (HPC) plays a key role in reducing hypoxia-induced apoptosis. We aimed to study the pharmacologic preconditioning effects of salidroside versus those of HPC in hypoxia-/anoxia-induced apoptosis in PC12 cells (pheochromocytoma). Material/Methods PC12 cells were treated by different experimental conditions: control condition, hypoxia condition, HPC condition, low-/middle-/high-dose condition of salidroside, cyclosporine A (CsA), and oratractyloside (ATR). The cell viability, lactate dehydrogenase (LDH) activity, apoptosis, mitochondrial membrane potential (MMP), intracellular Ca2+, caspase-3 activity, and expression of Bcl-2 were detected in PC12 cells after the hypoxia treatment. Salidroside, extracted from the traditional Chinese herb Rhodiola rosea L, plays an essential role in reducing hypoxia-induced apoptosis in PC12 cells by the mitochondrial pathway. Results Salidroside decreased the apoptosis and increased the viability of hypoxia-induced PC12 cells more effectively than HPC Moreover, salidroside markedly stabilized MMP and intracellular Ca2+, reduced or inhibited LDH and caspase-3 activity, and up-regulated Bcl-2; CsA and ATR showed corresponding function. Conclusions Salidroside administration restrains apoptosis induced by hypoxia in PC12 cells. The protective effects are mediated by preservation of mitochondrial integrity and MMP to inhibit the excessive Ca2+ influx and caspase-3 activity and to promote the Bcl-2 expression, providing a potential clinical and effective therapeutic mechanism to reduce deaths from ischemic or hypoxic injury. PMID:27794583

  13. In vitro effects of tea polyphenols on redox metabolism, oxidative stress, and apoptosis in PC12 cells.

    PubMed

    Raza, Haider; John, Annie

    2008-09-01

    Tea polyphenols, especially catechins, have been reported to be potent antioxidants and beneficial in oxidative stress-related diseases including cancer. Numerous animal and cell culture models demonstrate anticancer effects of tea catechins. Experimental and epidemiological evidence suggests the use of black tea polyphenols (BTP), green tea catechins (especially epigallocatechin gallate [EGCG]), and other polyphenols in preventing the progression of cancer both in animal and human populations. In the present study, we have demonstrated alterations in oxidative stress and redox metabolism using an isolated cell-free system and also in PC12 cancer cells after treatment with EGCG and BTP. We have demonstrated that tea catechins, alter the production of reactive oxygen species, glutathione metabolism, lipid peroxidation, and protein oxidation under in vitro conditions. We have also demonstrated that EGCG and BTP affect redox metabolism under cell culture conditions. Induction of apoptosis was observed, after the treatment with tea polyphenols, as shown by increased DNA breakdown and activation of the apoptotic markers, cytochrome c, caspase 3, and poly-(ADP-ribose) polymerase. These results may have implications in determining the chemopreventive and therapeutic use of tea catechins in vivo.

  14. Mitochondrial DNA damage and a hypoxic response are induced by CoCl2 in rat neuronal PC12 cells

    PubMed Central

    Wang, Guichun; Hazra, Tapas K.; Mitra, Sankar; Lee, Heung-Man; Englander, Ella W.

    2000-01-01

    Generation of reactive oxygen species (ROS) and activation of a transcriptional program that mimics the hypoxic response have been documented in cultured cells in the presence of cobalt chloride. We found that in the presence of hypoxia-mimicking concentrations of CoCl2, mitochondrial but not nuclear DNA damage is induced in rat neuronal, PC12 cells. To our knowledge, this is the first documentation of induction of mitochondrial DNA (mtDNA) damage under these conditions. Likewise, we provide the first evidence for elevation of MYH, the mammalian homolog of the Escherichia coli MutY DNA glycosylase, in mammalian cells. Recently, the human MYH was implicated in repair of oxidative DNA damage and shown to carry a mitochondrial localization sequence. Here, an induction of mtDNA damage and a time-dependent increase in the MYH level were detected with exposure of cells to 100 µM CoCl2. In addition, the levels of proteins involved in cellular responses to hypoxia, ROS and nuclear DNA damage; hypoxia-inducible factor 1α (HIF-1α), p53, p21 and PCNA were also modulated temporally. Earlier studies suggested that the mtDNA is a primary target for oxidative damage. Our findings extend these observations and suggest that activation of DNA repair processes is associated with the presence of mtDNA damage. PMID:10773083

  15. Mitochondrial DNA damage and a hypoxic response are induced by CoCl(2) in rat neuronal PC12 cells.

    PubMed

    Wang, G; Hazra, T K; Mitra, S; Lee, H M; Englander, E W

    2000-05-15

    Generation of reactive oxygen species (ROS) and activation of a transcriptional program that mimics the hypoxic response have been documented in cultured cells in the presence of cobalt chloride. We found that in the presence of hypoxia-mimicking concentrations of CoCl(2), mitochondrial but not nuclear DNA damage is induced in rat neuronal, PC12 cells. To our knowledge, this is the first documentation of induction of mitochondrial DNA (mtDNA) damage under these conditions. Likewise, we provide the first evidence for elevation of MYH, the mammalian homolog of the Escherichia coli MutY DNA glycosylase, in mammalian cells. Recently, the human MYH was implicated in repair of oxidative DNA damage and shown to carry a mitochondrial localization sequence. Here, an induction of mtDNA damage and a time-dependent increase in the MYH level were detected with exposure of cells to 100 microM CoCl(2). In addition, the levels of proteins involved in cellular responses to hypoxia, ROS and nuclear DNA damage; hypoxia-inducible factor 1alpha(HIF-1alpha), p53, p21 and PCNA were also modulated temporally. Earlier studies suggested that the mtDNA is a primary target for oxidative damage. Our findings extend these observations and suggest that activation of DNA repair processes is associated with the presence of mtDNA damage.

  16. Systemic Screening of Strains of the Lion's Mane Medicinal Mushroom Hericium erinaceus (Higher Basidiomycetes) and Its Protective Effects on Aβ-Triggered Neurotoxicity in PC12 Cells.

    PubMed

    Liu, Zongying; Wang, Qinglong; Cui, Jian; Wang, Lili; Xiong, Lili; Wang, Wei; Li, Diqiang; Liu, Na; Wu, Yiran; Mao, Canquan

    2015-01-01

    Hericium erinaceus possesses multiple medicinal values. To date, however, there have been few studies of the systemic screening of H. erinaceus strains, and the neuroprotective effects of H. erinaceus prepared from homogenized, fresh fruiting bodies are not fully understood. In this study, 4 random primers were selected and used in random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) to screen and evaluate the genetic diversity of 19 commercial strains of H. erinaceus from different localities in China. A total of 66 bands were obtained, and the percentage of polymorphic loci reached 80.30%. Five dendrograms were constructed based on RAPD by Jaccard cluster and within-group linkage analysis. Primer S20 as well as all 4 primers had great potential as specific primers for RAPD-PCR molecular identification and differentiation of H. erinaceus strains. Based on the results of submerged culture and fruiting body cultivation, strains HT-N, HT-J1, HT-C, and HT-M were identified as superior among the 19 H. erinaceus strains. Further study showed that the oral preparation of homogenized, fresh fruiting bodies of H. erinaceus could attenuate the Aβ25-35-triggered damage in PC12 cells by significantly increasing cell viability and by decreasing the release of lactate dehydrogenase. In conclusion, RAPD-PCR combined with liquid and solid cultures can be used well in the screening and identification of H. erinaceus strains, and products prepared from homogenized, fresh fruiting bodies of H. erinaceus had neuroprotective effects on PC12 cells.

  17. Novel fermented chickpea milk with enhanced level of γ-aminobutyric acid and neuroprotective effect on PC12 cells

    PubMed Central

    Li, Wen; Wei, Mingming; Wu, Junjun; Rui, Xin

    2016-01-01

    In this study, novel fermented chickpea milk with high γ -aminobutyric acid (GABA) content and potential neuroprotective activity was developed. Fermentation starter that can produce GABA was selected from 377 strains of lactic acid bacteria isolated from traditional Chinese fermented foods. Among the screened strains, strain M-6 showed the highest GABA-producing capacity in De Man–Rogosa and Sharp (MRS) broth and chickpea milk. M-6 was identified as Lactobacillus plantarum based on Gram staining, API carbohydrate fermentation pattern testing, and 16s rDNA sequencing. The complete gene encoding glutamate decarboxylase was cloned to confirm the presence of the gene in L. plantarum M-6. The fermentation condition was optimized by response surface methodology. Results demonstrated that L. plantarum M-6 produced the highest GABA content of 537.23 mg/L. The optimal condition included an inoculum concentration of 7%, presence of 0.2% (m/v) monosodium glutamate and 55 µ M pyridoxal-5-phosphate, incubation temperature of 39 °C and fermentation time of 48 h . GABA-enriched chickpea milk exerted protective effects on PC12 cells against MnCl2 -induced injury. GABA-enriched chickpea milk improved cell viability and markedly attenuated the release of lactate dehydrogenase compared with the impaired cells. PMID:27602272

  18. Modulatory effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells.

    PubMed

    Zhang, Min; Lee, Hak Ju; Park, Keun Hong; Park, Hyun Jin; Choi, Hyun Sook; Lim, Sung Cil; Lee, Myung Koo

    2012-06-01

    The effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Sesamin at concentration ranges of 20-75 μM exhibited a significant increase in intracellular dopamine levels at 24 h: 50 μM sesamin increased dopamine levels to 133% and tyrosine hydroxylase (TH) activity to 128.2% of control levels. Sesamin at 20-100 μM rapidly increased the intracellular levels of cyclic AMP (cAMP) to 158.3%-270.3% of control levels at 30 min. At 50 μM, sesamin combined with L-DOPA (50, 100 and 200 μM) further increased the intracellular dopamine levels for 24 h compared to L-DOPA alone. In the absence or presence of L-DOPA (100 and 200 μM), sesamin (50 μM) increased the phosphorylation of TH, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), as well as the mRNA levels of TH and CREB for 24 h, an effect which was reduced by L-DOPA (100 and 200 μM). In addition, 50 μM sesamin exhibited a protective effect against L-DOPA (100 and 200 μM)-induced cytotoxicity via the inhibition of reactive oxygen species (ROS) production and superoxide dismutase reduction, induction of extracellular signal-regulated kinase (ERK)1/2 and BadSer112 phosphorylation and Bcl-2 expression, and inhibition of cleaved-caspase-3 formation. These results suggested that sesamin enhanced dopamine biosynthesis and L-DOPA-induced increase in dopamine levels by inducing TH activity and TH gene expression, which was mediated by cAMP-PKA-CREB systems. Sesamin also protected against L-DOPA (100-200 μM)-induced cytotoxicity through the suppression of ROS activity via the modulation of ERK1/2, BadSer112, Bcl-2, and caspase-3 pathways in PC12 cells. Therefore, sesamin might serve as an adjuvant phytonutrient for neurodegenerative diseases.

  19. Down-regulation of hypoxia-inducible factor-2 in PC12 cells by nerve growth factor stimulation.

    PubMed

    Naranjo-Suárez, Salvador; Castellanos, María Carmen; Alvarez-Tejado, Miguel; Vara, Alicia; Landázuri, Manuel O; del Peso, Luis

    2003-08-22

    Cellular responses to low oxygen tension are mediated, at least in part, by the activation of the hypoxia-inducible factors (HIFs). In the presence of oxygen, specific HIF residues become hydroxylated by the action of a recently described group of dioxygenases. These post-translational modifications target HIF for proteosomal degradation and prevent its transcriptional activity. Despite these detailed studies, little is known about the regulation of HIF by stimuli other than hypoxia. Here we report that, in rat pheochromocytoma PC12 cells, nerve growth factor (NGF) stimulation results in a decrease of both basal and hypoxia-induced levels of HIF-2 alpha protein. NGF treatment did not increase HIF-hydroxylase gene expression or activity, and the reduction of the HIF-2 alpha protein level upon stimulation was observed even in the presence of HIF-hydroxylase inhibitors such as deferoxamine or dimethyloxoglutarate. Thus, in contrast to the response to hypoxia, the effect of NGF on HIF-2 alpha protein levels is not mediated by the HIF hydroxilases. Quantitative real time (RT)-PCR showed that NGF stimulation results in a decrease of the HIF-2 alpha mRNA level similar to that found at the protein level. Interestingly, NGF effect was specific for HIF-2 alpha mRNA because it did not affect HIF-1 alpha mRNA levels. NGF treatment reduced HIF-2 alpha mRNA levels even in the presence of actinomycin D, suggesting an effect on mRNA stability. Finally, the effect of NGF on HIF2 alpha correlates with reduction of both basal and hypoxia-induced vascular endothelial growth factor mRNA levels. Reporter assays suggest that the reduced expression of hypoxia-inducible genes upon NGF treatment is related, at least in part, to the reduction of HIF-2 alpha protein. Hence, in PC12 cells the level of HIF-2 alpha protein and its effect on gene expression can be down-regulated by stimuli other than oxygen.

  20. Common increase of GATA-3 level in PC-12 cells by three teratogens causing autism spectrum disorders.

    PubMed

    Rout, Ujjwal K; Clausen, Pete

    2009-06-01

    Autism spectrum disorder (ASD) is a disease of neuro-developmental origin of uncertain etiology. The current understanding is that both genetic and environmental factors contribute to the development of ASD. Exposure to valproate, thalidomide and alcohol during gestation are amongst the environmental triggers that are associated with the development of ASD. These teratogens may disturb the ontogeny of the brain by altering the expression pattern of genes that regulate the normal development of the brain. In this study, a neuron-like PC-12 cell model was used to examine the effects of these compounds on the binding potential of 50 different transcription factors to understand the molecular mechanism/s that may be involved in the teratogenesis caused by these agents. Cells in culture were treated with low or high concentrations of teratogens within a range that are reported in the blood of individuals. A pronounced increase in GATA transcription factor binding was observed for all three teratogens. Furthermore, Western blot analysis showed that GATA-3 level in the nuclear fractions was enhanced by each of the three teratogens. Results suggest that altered gene expression pattern due to heightened GATA-3 activities in the fetral brains following exposure to these teratogens may contribute to the development of ASD.

  1. Beta-asarone attenuates amyloid beta-induced autophagy via Akt/mTOR pathway in PC12 cells.

    PubMed

    Xue, Zhongfeng; Guo, Yalei; Zhang, Sheng; Huang, Liping; He, Yuping; Fang, Ruoming; Fang, Yongqi

    2014-10-15

    Alzheimer's disease (AD) is an age related and progressive neurodegenerative disease. Autophagy is a self-degradative process and plays a critical role in removing long-lived proteins and damaged organelles. Recent evidence suggests that autophagy might be involved in the pathogenesis of AD. β-asarone have various neuroprotective effects. However, the effect of β-asarone on autophagy in amyloid β-peptide (Aβ) induced cell injury is unclear, and little is known about the signaling pathway of β-asarone in autophagy regulation. The aim of the present study was to determine whether β-asarone protects cells from Aβ1-42 induced cytotoxicity via regulation of Beclin-1 dependent autophagy and its regulating signaling pathway. We examined effects of β-asarone on cell morphology, cell viability, neuron specific enolase (NSE) levels, autophagosomes and regulating Beclin-1, p-Akt and p-mTOR expressions in Aβ1-42 treated PC12 cells. We found that β-asarone could maintain the original morphology of cells and increase cell viability and decrease NSE levels significantly. Meanwhile, β-asarone decreased Beclin-1 expression significantly. In addition, β-asarone can increase levels of p-Akt and p-mTOR. These results showed that β-asarone protected cells from Aβ1-42 induced cytotoxicity and attenuated autophagy via activation of Akt-mTOR signaling pathway, which could be involved in neuroprotection of β-asarone against Aβ toxicity. Our findings suggest that β-asarone might be a potential preventive drug for AD.

  2. Overexpression of the human ubiquitin E3 ligase CUL4A alleviates hypoxia-reoxygenation injury in pheochromocytoma (PC12) cells

    SciTech Connect

    Tan, Can; Zhang, Li-Yang; Chen, Hong; Xiao, Ling; Liu, Xian-Peng; Zhang, Jian-Xiang

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Overexpression of human CUL4A (hCUL4A) in PC12 cells. Black-Right-Pointing-Pointer The effects of hCUL4A on hypoxia-reoxygenation injury were investigated. Black-Right-Pointing-Pointer hCUL4A suppresses apoptosis and DNA damage and thus promotes cell survival. Black-Right-Pointing-Pointer hCUL4A regulates apoptosis-related proteins and cell cycle regulators. -- Abstract: The ubiquitin E3 ligase CUL4A plays important roles in diverse cellular processes including carcinogenesis and proliferation. It has been reported that the expression of CUL4A can be induced by hypoxic-ischemic injury. However, the effect of elevated expression of CUL4A on hypoxia-reoxygenation injury is currently unclear. In this study, human CUL4A (hCUL4A) was expressed in rat pheochromocytoma (PC12) cells using adenoviral vector-mediated gene transfer, and the effects of hCUL4A expression on hypoxia-reoxygenation injury were investigated. In PC12 cells subjected to hypoxia and reoxygenation, we found that hCUL4A suppresses apoptosis and DNA damage by regulating apoptosis-related proteins and cell cycle regulators (Bcl-2, caspase-3, p53 and p27); consequently, hCUL4A promotes cell survival. Taken together, our results reveal the beneficial effects of hCUL4A in PC12 cells upon hypoxia-reoxygenation injury.

  3. Effect of alpha-ketoglutarate and N-acetyl cysteine on cyanide-induced oxidative stress mediated cell death in PC12 cells.

    PubMed

    Satpute, R M; Hariharakrishnan, J; Bhattacharya, R

    2010-06-01

    Cyanide is a mitochondrial poison, which is ubiquitously present in the environment. Cyanide-induced oxidative stress is known to play a key role in mediating the neurotoxicity and cell death in rat pheochromocytoma (PC12) cells. PC12 cells are widely used as a model for neurotoxicity assays in vitro. In the present study, we investigated the protective effects of alpha-ketoglutarate (A-KG), a potential cyanide antidote, and N-acetyl cysteine (NAC), an antioxidant against toxicity of cyanide in PC12 cells. Cells were treated with various concentrations (0.625-1.25 mM) of potassium cyanide (KCN) for 4 hours, in the presence or absence of simultaneous treatment of A-KG (0.5 mM) and NAC (0.25 mM). Cyanide caused marked decrease in the levels of cellular antioxidants like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). Lipid peroxidation indicated by elevated levels of malondialdehyde (MDA) was found to be accompanied by decreased levels of reduced glutathione (GSH) and total antioxidant status (TAS) of the cells. Cyanide-treated cells showed notable increase in caspase-3 activity and induction of apoptotic type of cell death after 24 hours. A-KG and NAC alone were very effective in restoring the levels of GSH and TAS, but together they significantly resolved the effects of cyanide on antioxidant enzymes, MDA levels, and caspase-3 activity. The present study reveals that combination of A-KG and NAC has critical role in abbrogating the oxidative stress-mediated toxicity of cyanide in PC12 cells. The results suggest potential role of A-KG and NAC in cyanide antagonism.

  4. Neferine attenuates the protein level and toxicity of mutant huntingtin in PC-12 cells via induction of autophagy.

    PubMed

    Wong, Vincent Kam Wai; Wu, An Guo; Wang, Jing Rong; Liu, Liang; Law, Betty Yuen-Kwan

    2015-02-18

    Mutant huntingtin aggregation is highly associated with the pathogenesis of Huntington's disease, an adult-onset autosomal dominant disorder, which leads to a loss of motor control and decline in cognitive function. Recent literature has revealed the protective role of autophagy in neurodegenerative diseases through degradation of mutant toxic proteins, including huntingtin or a-synuclein. Through the GFP-LC3 autophagy detection platform, we have  identified  neferine,  isolated  from  the  lotus  seed  embryo  of Nelumbo nucifera, which is able to induce autophagy through an AMPK-mTOR-dependent pathway. Furthermore, by overexpressing huntingtin with 74 CAG repeats (EGFP-HTT 74) in PC-12 cells, neferine reduces both the protein level and toxicity of mutant huntingtin through an autophagy-related gene 7 (Atg7)-dependent mechanism. With the variety of novel active compounds present in medicinal herbs, our current study suggests the possible protective mechanism of an autophagy inducer isolated from Chinese herbal medicine, which is crucial for its further development into a potential therapeutic agent for neurodegenerative disorders in the future.

  5. Activation of Nrf2 target enzymes conferring protection against oxidative stress in PC12 cells by ginger principal constituent 6-shogaol.

    PubMed

    Peng, Shoujiao; Yao, Juan; Liu, Yaping; Duan, Dongzhu; Zhang, Xiaolong; Fang, Jianguo

    2015-08-01

    Natural compounds containing phenoxyl groups and/or Michael acceptor units appear to possess antioxidant and cytoprotective properties. The ginger principal constituent 6-shogaol (6-S) represents one of such compounds. In this study, we reported that 6-S efficiently scavenges various free radicals in vitro, and displays remarkable cytoprotection against oxidative stress-induced cell damage in the neuron-like rat pheochromocytoma cell line, PC12 cells. Pretreatment of PC12 cells with 6-S significantly upregulates a series of phase II antioxidant molecules, such as glutathione, heme oxygenase 1, NAD(P)H: quinone oxidoreductase 1, thioredoxin reductase 1, and thioredoxin 1. A mechanistic study revealed that 6-S enhanced the translocation of Nrf2 from the cytosol to the nucleus and knockdown of Nrf2 abolished such protection, indicating that this cytoprotection is mediated by the activation of the transcription factor Nrf2. Another ginger constituent 6-gingerol (6-G), having a similar structure of 6-S but lacking the alpha,beta-unsaturated ketone structure (Michael acceptor moiety), failed to shelter PC12 cells from oxidative stress. Our results demonstrate that 6-S is a novel small molecule activator of Nrf2 in PC12 cells, and suggest that 6-S might be a potential candidate for the prevention of oxidative stress-mediated neurodegenerative disorders.

  6. Lignosus rhinocerotis (Cooke) Ryvarden mimics the neuritogenic activity of nerve growth factor via MEK/ERK1/2 signaling pathway in PC-12 cells

    PubMed Central

    Seow, Syntyche Ling-Sing; Eik, Lee-Fang; Naidu, Murali; David, Pamela; Wong, Kah-Hui; Sabaratnam, Vikineswary

    2015-01-01

    The traditional application of the sclerotium of Lignosus rhinocerotis (tiger’s milk mushroom) by the indigenous folks as tonic and remedy to treat a variety of ailments has been documented in Malaysia. Indigenous communities claimed to have consumed the decoction to boost their alertness during hunting. Mental alertness is believed to be related to neuronal health and neuroactivity. In the present study, the cell viability and neuritogenic effects of L. rhinocerotis sclerotium hot aqueous and ethanolic extracts, and crude polysaccharides on rat pheochromocytoma (PC-12) cells were studied. Interestingly, the hot aqueous extract exhibited neuritogenic activity comparable to NGF in PC-12 cells. However, the extracts and crude polysaccharides stimulated neuritogenesis without stimulating the production of NGF in PC-12 cells. The involvements of the TrkA receptor and MEK/ERK1/2 pathway in hot aqueous extract-stimulated neuritogenesis were examined by Trk (K252a) and MEK/ERK1/2 (U0126 and PD98059) inhibitors. There was no significant difference in protein expression in NGF- and hot aqueous extract-treated cells for both total and phosphorylated p44/42 MAPK. The neuritogenic activity in PC-12 cells stimulated by hot aqueous and ethanolic extracts, and crude polysaccharides of L. rhinocerotis sclerotium mimicking NGF activity via the MEK/ERK1/2 signaling pathway is reported for the first time. PMID:26542212

  7. Detection of the interaction between SNAP25 and rabphilin in neuroendocrine PC12 cells using the FLIM/FRET technique.

    PubMed

    Lee, Jiung-De; Chang, Yu-Fen; Kao, Fu-Jen; Kao, Lung-Sen; Lin, Chung-Chih; Lu, Ai-Chu; Shyu, Bai-Chuang; Chiou, Shih-Hwa; Yang, De-Ming

    2008-01-01

    Exocytosis has been proposed to contain four sequential steps, namely docking, priming, fusion, and recycling, and to be regulated by various proteins-protein interactions. Synaptosomal-associated protein of 25 kDa (SNAP25) has recently been found to bind rabphilin, the Rab3A specific binding protein, in vitro. However, it is still unclear whether SNAP25 and rabphilin interact during exocytosis within cells in vivo. This problem was addressed by the integration of fluorescence resonance energy transfer (FRET) with high sensitivity fluorescence lifetime imaging microscopy (FLIM) to observe this protein-protein interaction. Enhanced green fluorescence protein-labeled SNAP25 (donor) and red fluorescence protein-labeled rabphilin (acceptor) were expressed in neuroendocrine PC12 cells as a FRET pair and ATP stimulation was carried out for various durations. With 10 s stimulation, a 0.17-ns left shift of the lifetime peak was found when compared with donor only. Analysis of the lifetime image further suggested that the lifetime recovered to a similar level as the donor only in a time dependent manner. Four-dimensional (4D) images by FLIM provided useful information indicating that the interaction of SNAP25 and rabphilin occurred particularly within optical sections near cell membrane. Together the results suggest that SNAP25 bound rabphilin loosely at docking step before exocytosis and the binding became tighter at the very start of exocytosis. Finally, these two proteins dissociated after stimulation. To our knowledge, this is the first report to demonstrate the interaction of SNAP25 and rabphilin in situ using the FLIM-FRET technique within neuroendocrine cells.

  8. Ferulic acid prevents LPS-induced up-regulation of PDE4B and stimulates the cAMP/CREB signaling pathway in PC12 cells

    PubMed Central

    Huang, Hao; Hong, Qian; Tan, Hong-ling; Xiao, Cheng-rong; Gao, Yue

    2016-01-01

    Aim: Phosphodiesterase 4 (PDE4) isozymes are involved in different functions, depending on their patterns of distribution in the brain. The PDE4 subtypes are distributed in different inflammatory cells, and appear to be important regulators of inflammatory processes. In this study we examined the effects of ferulic acid (FA), a plant component with strong anti-oxidant and anti-inflammatory activities, on lipopolysaccharide (LPS)-induced up-regulation of phosphodiesterase 4B (PDE4B) in PC12 cells, which in turn regulated cellular cAMP levels and the cAMP/cAMP response element binding protein (CREB) pathway in the cells. Methods: PC12 cells were treated with LPS (1 μg/mL) for 8 h, and the changes of F-actin were detected using laser scanning confocal microscopy. The levels of pro-inflammatory cytokines were measured suing ELISA kits, and PDE4B-specific enzymatic activity was assessed with a PDE4B assay kit. The mRNA levels of PDE4B were analyzed with Q-PCR, and the protein levels of CREB and phosphorylated CREB (pCREB) were determined using immunoblotting. Furthermore, molecular docking was used to identify the interaction between PDE4B2 and FA. Results: Treatment of PC12 cells with LPS induced thick bundles of actin filaments appearing in the F-actin cytoskeleton, which were ameliorated by pretreatment with FA (10–40 μmol/L) or with a PDE4B inhibitor rolipram (30 μmol/L). Pretreatment with FA dose-dependently inhibited the LPS-induced production of TNF-α and IL-1β in PC12 cells. Furthermore, pretreatment with FA dose-dependently attenuated the LPS-induced up-regulation of PDE4 activity in PC12 cells. Moreover, pretreatment with FA decreased LPS-induced up-regulation of the PDE4B mRNA, and reversed LPS-induced down-regulation of CREB and pCREB in PC12 cells. The molecular docking results revealed electrostatic and hydrophobic interactions between FA and PDE4B2. Conclusion: The beneficial effects of FA in PC12 cells might be conferred through inhibition of LPS

  9. NFAT1 and NFAT3 Cooperate with HDAC4 during Regulation of Alternative Splicing of PMCA Isoforms in PC12 Cells

    PubMed Central

    Kosiorek, Michalina; Podszywalow-Bartnicka, Paulina; Zylinska, Ludmila; Pikula, Slawomir

    2014-01-01

    Background The bulk of human genes undergo alternative splicing (AS) upon response to physiological stimuli. AS is a great source of protein diversity and biological processes and is associated with the development of many diseases. Pheochromocytoma is a neuroendocrine tumor, characterized by an excessive Ca2+-dependent secretion of catecholamines. This underlines the importance of balanced control of calcium transport via regulation of gene expression pattern, including different calcium transport systems, such as plasma membrane Ca2+-ATPases (PMCAs), abundantly expressed in pheochromocytoma chromaffin cells (PC12 cells). PMCAs are encoded by four genes (Atp2b1, Atp2b2, Atp2b3, Atp2b4), whose transcript products undergo alternative splicing giving almost 30 variants. Results In this scientific report, we propose a novel mechanism of regulation of PMCA alternative splicing in PC12 cells through cooperation of the nuclear factor of activated T-cells (NFAT) and histone deacetylases (HDACs). Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA. RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells. NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well. Furthermore, chromatin immunoprecipitation experiments showed that NFAT1-HDAC4 or NFAT3-HDAC4 complexes might be involved in regulation of PMCA2x splicing variant generation. Conclusions We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells. PMID:24905014

  10. Neurite Outgrowth in PC12 Cells Stimulated by Components from Dendranthema × grandiflorum cv. “Mottenohoka” Is Enhanced by Suppressing Phosphorylation of p38MAPK

    PubMed Central

    Kimura, Hirokazu; Tsukagoshi, Hiroyuki; Kozawa, Kunihisa; Koketsu, Mamoru; Ninomiya, Masayuki; Furukawa, Shoei

    2013-01-01

    Components from Dendranthema × grandiflorum cv. “Mottenohoka” that promote neurite outgrowth of PC12 cells were identified and the mechanism of neurite outgrowth stimulated by isolated components was studied. Components that promoted the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) of PC12 cells were isolated. From various structural analyses, the active components were identified as acacetin and luteolin. The effects of acacetin or luteolin on PC12 cells were evaluated by electro-blotting and immunostaining. Slight neurite outgrowth in PC12 cells was observed within 2 days of culture after stimulation by luteolin or acacetin. However, NGF-stimulation induced remarkable neurite outgrowth in comparison. Neurite outgrowth by luteolin or acacetin was significantly enhanced by pretreatment with SB203580 (a p38MAPK inhibitor). The results of this study into the phosphorylation of ERK 1/2 and p38MAPK by flavonoids suggest that the inhibition of p38MAPK phosphorylation may effectively enhance neurite outgrowth. PMID:23554829

  11. Hypoxia pretreatment and EPO-modification enhance the protective effects of MSC on neuron-like PC12 cells in a similar way.

    PubMed

    Feng, Jinli; Wang, Wei

    2017-01-08

    Mesenchymal stem cells (MSC) based cell transplantation therapy is proved to be an attractive strategy with great potential for improvement of hypoxia induced neural damage. In the present study, MSCs were co-culture with PC12 to investigate its protective effects against hypoxia pretreatment, and the Lactate dehydrogenase (LDH) release assay, MTT and Anexin V staining were performed to analysis the cellular damage or apoptotic. RT-PCR and Western blotting were further used to investigate the underlying mechanism. The results indicate that hypoxia treatment results in the decrease of PC12 cell viability, yet co-culture with MSC could protect the PC12 from hypoxia induced damage. Hypoxia pre-activated or EPO transduced MSC with up-regulated erythropoietin (EPO) expression could further enhance MSC's protective effect against hypoxia induced cell damage, which was associated with high level of anti-apoptotic p-Akt and ration Bcl-2/Bax, and decreased Caspase 3 in PC12. Taken together, these data suggests high levels of MSC-mediated cyto-protection is closely tied to high gene expression levels of EPO. The up-regulation of EPO for enhanced MSC-mediated cyto-protection may has great potential for the MSC cellular therapy of neural or neuronal injuries induced by hypoxia.

  12. Role of the bZIP transcription factor IREBF1 in the NGF induction of stromelysin-1 (transin) gene expression in PC12 cells.

    PubMed

    deSouza, S; Nordstrom, L A; Ciment, G

    1997-06-01

    Stromelysin-1 (ST-1) is one of the most nerve growth factor-(NGF) responsive gene products expressed in PC12 cells. In previous work, we identified a novel NGF-responsive element in the proximal promoter region of the ST-1 gene that participates in this induction, and showed that it bound a protein present in the nuclei of PC12 cells. Here, we identify a transcription factor that specifically recognizes this regulatory element-the interferon-response element binding factor-1 (IREBF1), a member of the basic leucine zipper gene family. We show that IREBF1 is constitutively expressed in PC12 cells and that overexpression of IREBF1 augments NGF-responsive ST-1 gene regulation, but does not affect basal levels of expression. On the other hand, expression of a mutated form of this transcription factor lacking the DNA binding domain attenuated NGF responsiveness, without affecting basal levels of expression. These data suggest that IREBF1 is part of the NGF-responsive transcriptional machinery necessary for the expression of ST-1 in PC12 cells.

  13. Dual actions of lindane ({gamma}-hexachlorocyclohexane) on calcium homeostasis and exocytosis in rat PC12 cells

    SciTech Connect

    Heusinkveld, Harm J.; Thomas, Gareth O.; Lamot, Ischa; Berg, Martin van den; Kroese, Alfons B.A.; Westerink, Remco H.S.

    2010-10-01

    The persistent organochlorine pesticide lindane is still abundantly found in the environment and in human and animal tissue samples. Lindane induces a wide range of adverse health effects, which are at least partially mediated via the known inhibition of GABA{sub A} and glycine receptors. Additionally, lindane has been reported to increase the basal intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}). As Ca{sup 2+} triggers many cellular processes, including cell death and vesicular neurotransmitter release (exocytosis), we investigated whether lindane affects exocytosis, Ca{sup 2+} homeostasis, production of reactive oxygen species (ROS) and cytotoxicity in neuroendocrine PC12 cells. Amperometric recordings and [Ca{sup 2+}]{sub i} imaging experiments with fura-2 demonstrated that lindane ({>=} 10 {mu}M) rapidly increases basal exocytosis and basal [Ca{sup 2+}]{sub i}. Additional imaging and electrophysiological recordings revealed that this increase was largely due to a lindane-induced membrane depolarization and subsequent opening of N- and P/Q-type voltage-gated Ca{sup 2+} channels (VGCC). On the other hand, lindane ({>=} 3 {mu}M) induced a concentration-dependent but non-specific inhibition of VGCCs, thereby limiting the lindane-induced increase in basal [Ca{sup 2+}]{sub i} and exocytosis. Importantly, the non-specific inhibition of VGCCs also reduced stimulation-evoked exocytosis and Ca{sup 2+} influx. Though lindane exposure concentration-dependently increased ROS production, cell viability was not affected indicating that the used concentrations were not acute cytotoxic. These combined findings indicate that lindane has two, partly counteracting effects. Lindane causes membrane depolarization, thereby increasing basal [Ca{sup 2+}]{sub i} and exocytosis. In parallel, lindane inhibits VGCCs, thereby limiting the basal effects and reducing stimulation-evoked [Ca{sup 2+}]{sub i} and exocytosis. This study further underlines the need to consider

  14. Modification of HSP proteins and Ca2+ are responsible for the NO-derived peroxynitrite mediated neurological damage in PC12 cell

    PubMed Central

    Wen, Jun; Li, Hua; Zhang, Yudan; Li, Xia; Liu, Fang

    2015-01-01

    Peroxynitrite as one crucial metabolite of NO-derived agents has been well multi-investigated to inspect its potential role and sought to define its concrete mechanism underlying the memory loss and impaired cognition involved in pathological processes. In this investigation, the cell viability was assessed by the MTT assay. The neurotoxicity of peroxynitrite was analyzed by using immunohistochemical measurements in cultured PC12 cells to explore the underlying mechanisms. The generation of ROS was evaluated by a fluorometry assay by a fluorometry assay. Apoptosis was assayed by annexin V-FITC and PI staining with flow cytometry. [Ca2+]i was examined by using the microspectrofluorometer. Hsp70 was detected by western blot assay. The results revealed that PC12 cells were inhibited by peroxynitrite both in a dose-dependent and time-dependent manner. The level of ROS in PC12 cells exposed to SIN-1 was increased in a dose-dependent manner. The result indicated that the SIN-1 induced apoptosis of PC12 cells in a dose-dependent manner. Quercetin inhibited the viability of PC12 cells in a concentration-dependent manner. [Ca2+]i was increased gradually when cells treated with quercetin alone and also increased with treatment of dantrolene-containing. Hsp70 was significantly decreased in SIN-1-treated group compared with that of control group (P<0.01). In conclusion, Ca2+ homeostasis and chaperone Hsp70 were critically involved in peroxynitrite induced nitrosative stress as protective. Peroxynitrite acts as the pathological agent in learning and memory defects in CNS disorders associated with challenge. PMID:26191139

  15. Modification of HSP proteins and Ca2+ are responsible for the NO-derived peroxynitrite mediated neurological damage in PC12 cell.

    PubMed

    Wen, Jun; Li, Hua; Zhang, Yudan; Li, Xia; Liu, Fang

    2015-01-01

    Peroxynitrite as one crucial metabolite of NO-derived agents has been well multi-investigated to inspect its potential role and sought to define its concrete mechanism underlying the memory loss and impaired cognition involved in pathological processes. In this investigation, the cell viability was assessed by the MTT assay. The neurotoxicity of peroxynitrite was analyzed by using immunohistochemical measurements in cultured PC12 cells to explore the underlying mechanisms. The generation of ROS was evaluated by a fluorometry assay by a fluorometry assay. Apoptosis was assayed by annexin V-FITC and PI staining with flow cytometry. [Ca2+]i was examined by using the microspectrofluorometer. Hsp70 was detected by western blot assay. The results revealed that PC12 cells were inhibited by peroxynitrite both in a dose-dependent and time-dependent manner. The level of ROS in PC12 cells exposed to SIN-1 was increased in a dose-dependent manner. The result indicated that the SIN-1 induced apoptosis of PC12 cells in a dose-dependent manner. Quercetin inhibited the viability of PC12 cells in a concentration-dependent manner. [Ca2+]i was increased gradually when cells treated with quercetin alone and also increased with treatment of dantrolene-containing. Hsp70 was significantly decreased in SIN-1-treated group compared with that of control group (P<0.01). In conclusion, Ca2+ homeostasis and chaperone Hsp70 were critically involved in peroxynitrite induced nitrosative stress as protective. Peroxynitrite acts as the pathological agent in learning and memory defects in CNS disorders associated with challenge.

  16. MiR-21 is an Ngf-modulated microRNA that supports Ngf signaling and regulates neuronal degeneration in PC12 cells.

    PubMed

    Montalban, Enrica; Mattugini, Nicola; Ciarapica, Roberta; Provenzano, Claudia; Savino, Mauro; Scagnoli, Fiorella; Prosperini, Gianluca; Carissimi, Claudia; Fulci, Valerio; Matrone, Carmela; Calissano, Pietro; Nasi, Sergio

    2014-06-01

    The neurotrophins Ngf, Bdnf, NT-3, NT4-5 have key roles in development, survival, and plasticity of neuronal cells. Their action involves broad gene expression changes at the level of transcription and translation. MicroRNAs (miRs)-small RNA molecules that control gene expression post-transcriptionally-are increasingly implicated in regulating development and plasticity of neural cells. Using PC12 cells as a model system, we show that Ngf modulates changes in expression of a variety of microRNAs, including miRs known to be modulated by neurotrophins-such as the miR-212/132 cluster-and several others, such as miR-21, miR-29c, miR-30c, miR-93, miR-103, miR-207, miR-691, and miR-709. Pathway analysis indicates that Ngf-modulated miRs may regulate many protein components of signaling pathways involved in neuronal development and disease. In particular, we show that miR-21 enhances neurotrophin signaling and controls neuronal differentiation induced by Ngf. Notably, in a situation mimicking neurodegeneration-differentiated neurons deprived of Ngf-this microRNA is able to preserve the neurite network and to support viability of the neurons. These findings uncover a broad role of microRNAs in regulating neurotrophin signaling and suggest that aberrant expression of one or more Ngf-modulated miRs may be involved in neurodegenerative diseases.

  17. Butyl benzyl phthalate blocks Ca{sup 2+} signaling coupled with purinoceptor in rat PC12 cells

    SciTech Connect

    Liu, P.-S. . E-mail: psliu@mail.scu.edu.tw; Chen, Y.-Y.

    2006-01-15

    Butyl benzyl phthalate (BBP) is a plasticizer and causes public concern because of its genomic estrogenic effects via estrogen receptors. We previously found that BBP has non-genomic effects, exerting inhibitory effects on the functional activities of nicotinic acetylcholine receptors (nAChR) in bovine adrenal chromaffin cells. nAChR belongs to the superfamily of neurotransmitter-gated channels, so does P2X purinoceptor that is widely distributed in the nervous system and play a role in pain reactions. In this study, we investigated the effects of BBP on the change of [Ca{sup 2+}]{sub c} (cytosolic calcium ion concentration) under the stimulation of purinoceptors in PC12 cells and found that BBP inhibited ATP-induced [Ca{sup 2+}]{sub c} rise (IC{sub 5} = 8.3 {mu}M). The inhibitory rate of BBP remained under the increase of ATP concentration; therefore, the possibility of competitive inhibition was excluded. The inhibition of BBP on P2Y was excluded because its inhibition on ATP-induced [Ca{sup 2+}]{sub c} rise was not found in the absence of extracellular Ca{sup 2+}. BBP might have some actions on voltage-operated Ca{sup 2+} channels (VOCCs) since BBP inhibited the Ca{sup 2+} signaling responding to high K{sup +} stimulation (IC{sub 5} = 1.2 {mu}M). We suggest that BBP inhibits the ATP-induced [Ca{sup 2+}]{sub c} rise via its non-competitive inhibition on P2X purinoceptors and VOCCs in the plasma membrane.

  18. Is the PentaBDE Replacement, Tris (1,3-dichloro-2-propyl) Phosphate (TDCPP), a Developmental Neurotoxicant? Studies in PC12 Cells

    PubMed Central

    Dishaw, Laura V.; Powers, Christina M.; Ryde, Ian T.; Roberts, Simon C.; Seidler, Frederic J.; Slotkin, Theodore A.; Stapleton, Heather M.

    2011-01-01

    Organophosphate flame retardants (OPFRs) are used as replacements for the commercial PentaBDE mixture that was phased out in 2004. OPFRs are ubiquitous in the environment and detected at high concentrations in residential dust, suggesting widespread human exposure. OPFRs are structurally similar to neurotoxic organophosphate pesticides, raising concerns about exposure and toxicity to humans. This study evaluated the neurotoxicity of tris (1,3-dichloro-2-propyl) phosphate (TDCPP) compared to the organophosphate pesticide, chlorpyrifos (CPF), a known developmental neurotoxicant. We also tested the neurotoxicity of three structurally similar OPFRs, tris (2-chloroethyl) phosphate (TCEP), tris (1-chloropropyl) phosphate (TCPP), and tris (1,3-dibromopropyl) phosphate (TDBPP), and 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), a major component of PentaBDE. Using undifferentiated and differentiating PC12 cells, changes in DNA synthesis, oxidative stress, differentiation into dopaminergic or cholinergic neurophenotypes, cell number, cell growth and neurite growth were assessed. TDCPP displayed concentration-dependent neurotoxicity, often with effects equivalent to or greater than equimolar concentrations of CPF. TDCPP inhibited DNA synthesis, and all OPFRs decreased cell number, and altered neurodifferentiation. Although TDCPP elevated oxidative stress, there was no adverse effect on cell viability or growth. TDCPP and TDBPP promoted differentiation into both neuronal phenotypes, while TCEP and TCPP promoted only the cholinergic phenotype. BDE-47 had no effect on cell number, cell growth or neurite growth. Our results demonstrate that different OPFRs show divergent effects on neurodifferentiation, suggesting the participation of multiple mechanisms of toxicity. Additionally, these data suggest that OPFRs may affect neurodevelopment with similar or greater potency compared to known and suspected neurotoxicants. PMID:21255595

  19. Is the PentaBDE replacement, tris (1,3-dichloro-2-propyl) phosphate (TDCPP), a developmental neurotoxicant? Studies in PC12 cells

    SciTech Connect

    Dishaw, Laura V.; Powers, Christina M.; Ryde, Ian T.; Roberts, Simon C.; Seidler, Frederic J.; Slotkin, Theodore A.; Stapleton, Heather M.

    2011-11-15

    Organophosphate flame retardants (OPFRs) are used as replacements for the commercial PentaBDE mixture that was phased out in 2004. OPFRs are ubiquitous in the environment and detected at high concentrations in residential dust, suggesting widespread human exposure. OPFRs are structurally similar to neurotoxic organophosphate pesticides, raising concerns about exposure and toxicity to humans. This study evaluated the neurotoxicity of tris (1,3-dichloro-2-propyl) phosphate (TDCPP) compared to the organophosphate pesticide, chlorpyrifos (CPF), a known developmental neurotoxicant. We also tested the neurotoxicity of three structurally similar OPFRs, tris (2-chloroethyl) phosphate (TCEP), tris (1-chloropropyl) phosphate (TCPP), and tris (2,3-dibromopropyl) phosphate (TDBPP), and 2,2 Prime ,4,4 Prime -tetrabromodiphenyl ether (BDE-47), a major component of PentaBDE. Using undifferentiated and differentiating PC12 cells, changes in DNA synthesis, oxidative stress, differentiation into dopaminergic or cholinergic neurophenotypes, cell number, cell growth and neurite growth were assessed. TDCPP displayed concentration-dependent neurotoxicity, often with effects equivalent to or greater than equimolar concentrations of CPF. TDCPP inhibited DNA synthesis, and all OPFRs decreased cell number and altered neurodifferentiation. Although TDCPP elevated oxidative stress, there was no adverse effect on cell viability or growth. TDCPP and TDBPP promoted differentiation into both neuronal phenotypes, while TCEP and TCPP promoted only the cholinergic phenotype. BDE-47 had no effect on cell number, cell growth or neurite growth. Our results demonstrate that different OPFRs show divergent effects on neurodifferentiation, suggesting the participation of multiple mechanisms of toxicity. Additionally, these data suggest that OPFRs may affect neurodevelopment with similar or greater potency compared to known and suspected neurotoxicants.

  20. H2S protects PC12 cells against toxicity of corticosterone by modulation of BDNF-TrkB pathway.

    PubMed

    Gao, Shenglan; Li, Wenting; Zou, Wei; Zhang, Ping; Tian, Ying; Xiao, Fan; Gu, Hongfeng; Tang, Xiaoqing

    2015-11-01

    Corticosterone, one of the glucocorticoids, is toxic to neurons and plays an important role in depressive-like behavior and depression. We previously showed that hydrogen sulfide (H2S), a novel physiological mediator, plays an inhibitory role in depression. However, the mechanism underlying H2S-triggered antidepressant-like role is not clearly known. Brain-derived neurotrophic factor (BDNF), a neurotrophic factor, plays a neuroprotective role that is mediated by its high-affinity tropomysin-related kinase B (TrkB) receptor. In this study, to investigate the underlying mechanism of H2S-induced antidepressant-like role, we explored whether H2S could protect neurons against corticosterone-mediated cyctotoxicity and whether this protective role of H2S was involved in the regulation of BDNF-TrkB pathway. Our data demonstrated that sodium hydrosulfide (NaHS), the donor of H2S, could prevent corticosterone-induced cytotoxicity, apoptosis, accumulation of intracellular reactive oxygen species (ROS) and loss of mitochondrial membrane potential (MMP) in PC12 cells. NaHS not only induced the up-regulation of BDNF but also prevented the down-regulation of BDNF by corticosterone. It was also found that blocking BDNF-TrkB pathway by K252a, an inhibitor of TrkB, abolished the protection of H2S against corticosterone-induced cytotoxicity, apoptosis, accumulation of ROS, and loss of MMP. These results suggest that H2S protects against the neurotoxicity of corticosterone by modulation of the BDNF-TrkB pathway.

  1. Downregulation of Mfn2 participates in manganese-induced neuronal apoptosis in rat striatum and PC12 cells.

    PubMed

    Liu, Xinhang; Yang, Jianbin; Lu, Chunhua; Jiang, Shengyang; Nie, Xiaoke; Han, Jingling; Yin, Lifeng; Jiang, Junkang

    2017-02-21

    Manganese (Mn) is a widely distributed trace element that is essential for normal brain function and development. However, chronic exposure to excessive Mn has been known to lead to neuronal loss and manganism, a disease with debilitating motor and cognitive deficits, whose clinical syndrome resembling idiopathic Parkinson's disease (IPD). However, the precise molecular mechanism underlying Mn neurotoxicity remains largely unclear. Accumulating evidence indicates that abnormal mitochondrial functionality is an early and causal event in Mn-induced neurodegeneration and apoptosis. Here, we investigated whether Mitofusin 2 (Mfn2), a highly conserved dynamin-related protein (DRP), played a role in the regulation of Mn-induced neuronal apoptosis. We revealed that Mfn2 was significantly dysregulated in rat striatum and PC12 neuronal-like cells following Mn exposure. Western blot analysis revealed that the expression of Mfn2 was remarkably decreased following different concentrations of Mn exposure. Immunohistochemistry analysis confirmed a remarkable downregulation of Mfn2 in rat striatum after Mn exposure. Immunofluorescent staining showed that Mfn2 was expressed predominantly in neurons, and neuronal loss of Mfn2 was associated with the expression of active caspase-3 following Mn exposure. Importantly, overexpression of Mfn2 apparently attenuated Mn-induced neuronal apoptosis. Notably, treatment with caspase-3 inhibitor Ac-DEVD-CH could not rescue Mn-induced downregulation of Mfn2, suggesting that Mn-induced mfn2 occurs prior to neuronal apoptosis. Taken together, these results indicated that down-regulated expression of Mfn2 might contribute to the pathological processes underlying Mn neurotoxicity.

  2. The activity of cAMP-dependent protein kinase is required at a posttranslational level for induction of voltage-dependent sodium channels by peptide growth factors in PC12 cells

    PubMed Central

    1992-01-01

    The synthesis and expression of voltage-dependent sodium (Na) channels is a crucial aspect of neuronal differentiation because of the central role these ion channels play in the generation of action potentials and the transfer of information in the nervous system. We have used rat pheochromocytoma (PC12) cell lines deficient in cAMP-dependent protein kinase (PKA) activity to examine the role of PKA in the induction of Na channel expression by nerve growth factor (NGF) and basic FGF (bFGF). In the parental PC12 cell line both NGF and bFGF elicit an increase in the density of functional Na channels, as determined from whole-cell patch clamp recordings. This increase does not occur in two PC12 cell lines deficient in both isozymes of PKA (PKAI and PKAII), and is strongly reduced in a third line deficient in PKAII, but not PKAI. Despite the inability of the neurotrophic factors to induce functional Na channel expression in the PKA-deficient cells, Northern blot hybridization studies and saxitoxin binding assays of intact cells indicate that NGF and bFGF are still capable of eliciting increases in both Na channel mRNA and Na channel protein in the membrane. Thus, PKA activity appears to be necessary at a posttranslational step in the synthesis and expression of functional Na channels, and thereby plays an important role in determining neuronal excitability. PMID:1311713

  3. [EFFECT OF FUCOIDANS ISOLATED FROM SEAWEEDS LAMINARIA DIGITATA AND FUCUS VESICULOSUS ON CELL LINES HELA G-63, ECV 304 AND PC 12].

    PubMed

    Zhurishkina, E V; Lapina, I M; Ivanen, D R; Stepanov, S I; Shvetsova, S V; Shavarda, A L; Giliano, N Ya; Kulminskaya, A A

    2015-01-01

    The aim of the research was to investigate cytotoxicity of fucoidans on mammals cells. Three different samples of fucoidans were isolated from mechanically grounded brown algae Laminaria digitata and Fucus ve- siculosus. The sample F2 that differed from the others by higher sulfatation level and suppression of HeLa G-63 line culture growth was taken for further study in cell lines HeLa G-63, ECV 304 and PC 12. We have shown that fucoidan preparation F2 inhibits proliferation and induces cell death in a dose- and time-dependent manner for all investigated cell lines. Neuroendocrine tumor rat cell line PC 12 appeared to be the most sensitive to fucoidan treatment whereas endothelial human cells ECV 304 were the least sensitive.

  4. Protective Effect of 25Mg-Porphyrin-Fullerene Nanoparticles on Oxygen-Glucose Deprivation/Reperfusion Injury in PC12 Cells.

    PubMed

    Ghasemzadeh, Peivand; Rezayat, Seyed Mahdi; Adeli, Sharareh; Rahbar-Roshandel, Nahid

    2016-08-01

    We investigated the effects of 25Mg-Porphyrin-Fullerene nanoparticles, (25MgPMC16) smart ferroporphyrin nanoparticles, on PC12 cells exposed to oxygen-glucose deprivation/reperfusion. In order to explore its effect on cells under oxygen-glucose deprivation conditions, the cultures were pretreated with 25MgPMC16 24 hours prior to oxygen-glucose deprivation/reperfusion. To initiate the oxygen-glucose deprivation/reperfusion, the cell culture medium was replaced with a glucose-free medium and the cells were transferred to a humidified incubation chamber in a mixture of 95% N2 and 5% CO2 at 37° C for 30, 60 and 120 min. Cell viability was assessed by MTT assay. Exposure of PC12 cells to 30, 60 and 120 min oxygen-glucose deprivation significantly decreased the cell viability. Pretreatment of the cultures with 25MgPMC16 significantly increased cell viability in a concentration-dependent manner. Pretreatment, the cultures with MK-801 (10 µM), a non-competitive NMDA antagonist, has attenuated the cell death after 30 min oxygen-glucose deprivation. We concluded that 25MgPMC16 could protect PC12 cells against oxygen-glucose deprivation/reperfusion-induced cell injury in a concentration-dependent manner. That could be due to the effect of 25MgPMC16 on ATP synthesis and the antioxidant effects of its components.

  5. A cAMP-Dependent, Protein Kinase A-Independent Signaling Pathway Mediating Neuritogenesis through Egr1 in PC12 Cells

    PubMed Central

    Ravni, Aurélia; Vaudry, David; Gerdin, Matthew J.; Eiden, Maribeth V.; Falluel-Morel, Anthony; Gonzalez, Bruno J.; Vaudry, Hubert; Eiden, Lee E.

    2014-01-01

    The neurotrophic peptide PACAP (pituitary adenylate cyclase-activating polypeptide) elevates cAMP in PC12 cells. Forskolin and dibutyryl cAMP mimic PACAP’s neuritogenic and cell morphological effects, suggesting that they are driven by cAMP. Comparison of microarray expression profiles after exposure of PC12 cells to either forskolin, dibutyryl cAMP, or PACAP revealed a small group of cAMP-dependent target genes. Neuritogenesis induced by all three agents is protein kinase A (PKA)-independent [not blocked by N-[2-(4-bromocinnamylamino)ethyl]-5-isoquino-line (H89)] and extracellular signal-regulated kinase (ERK)-dependent [blocked by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio) butadiene (U0126)], and therefore cAMP-dependent target genes potentially mediating neuritogenesis were selected for further analysis based on the pharmacological profile of their induction by PACAP (i.e., mimicking that of neuritogenesis). Small interfering RNA (siRNA) targeting one of these genes, Egr1, blocked PACAP-induced neuritogenesis, and siRNA targeting another, Vil2, blocked a component of the cell size increase elicited by PACAP. Neither siRNA blocked PACAP’s PKA-dependent antiproliferative effects. PACAP signaling to neuritogenesis was also impaired by dominant-negative Rap1 expression but was not affected by inhibition of protein kinase C (PKC), indicating a G-protein-coupled receptor-mediated differentiation pathway distinct from the one activated by receptor tyrosine kinase ligands such as nerve growth factor (NGF), that involves both Rap1 and PKC. We have thus identified a cAMP-dependent, PKA-independent pathway proceeding through ERK that functions to up-regulate the transcription of two genes, Egr1 and Vil2, required for PACAP-dependent neuritogenesis and increased cell size, respectively. Dominant-negative Rap1 expression impairs both PACAP-induced neuritogenesis and Egr1 activation by PACAP, suggesting that cAMP elevation and ERK activation by PACAP are linked through

  6. Neurite outgrowth stimulatory effects of myco synthesized AuNPs from Hericium erinaceus (Bull.: Fr.) Pers. on pheochromocytoma (PC-12) cells

    PubMed Central

    Raman, Jegadeesh; Lakshmanan, Hariprasath; John, Priscilla A; Zhijian, Chan; Periasamy, Vengadesh; David, Pamela; Naidu, Murali; Sabaratnam, Vikineswary

    2015-01-01

    Background Hericium erinaceus has been reported to have a wide range of medicinal properties such as stimulation of neurite outgrowth, promotion of functional recovery of axonotmetic peroneal nerve injury, antioxidant, antihypertensive, and antidiabetic properties. In recent years, the green synthesis of gold nanoparticles (AuNPs) has attracted intense interest due to the potential use in biomedical applications. The aim of this study was to investigate the effects of AuNPs from aqueous extract of H. erinaceus on neurite outgrowth of rat pheochromocytoma (PC-12) cells. Methods The formation of AuNPs was characterized by UV–visible spectrum, energy dispersive X-ray (EDX), field-emission scanning electron microscope (FESEM), transmission electron microscopy (TEM), particle size distribution, and Fourier transform-infrared spectroscopy (FTIR). Furthermore, the neurite extension study of synthesized AuNPs was evaluated by in vitro assay. Results The AuNPs exhibited maximum absorbance between 510 and 600 nm in UV–visible spectrum. FESEM and TEM images showed the existence of nanoparticles with sizes of 20–40 nm. FTIR measurements were carried out to identify the possible biomolecules responsible for capping and efficient stabilization of the nanoparticles. The purity and the crystalline properties were confirmed by EDX diffraction analysis, which showed strong signals with energy peaks in the range of 2–2.4 keV, indicating the existence of gold atoms. The synthesized AuNPs showed significant neurite extension on PC-12 cells. Nerve growth factor 50 ng/mL was used as a positive control. Treatment with different concentrations (nanograms) of AuNPs resulted in neuronal differentiation and neuronal elongation. AuNPs induced maximum neurite outgrowth of 13% at 600 ng/mL concentration. Conclusion In this study, the AuNPs synthesis was achieved by a simple, low-cost, and rapid bioreduction approach. AuNPs were shown to have potential neuronal differentiation and

  7. Preconditioning with Gua Lou Gui Zhi decoction enhances H2O2-induced Nrf2/HO-1 activation in PC12 cells.

    PubMed

    Mao, Jingjie; Li, Zuanfang; Lin, Ruhui; Zhu, Xiaoqin; Lin, Jiumao; Peng, Jun; Chen, Lidian

    2015-09-01

    Spasticity is common in various central neurological conditions, including after a stroke. Such spasticity may cause additional problems, and often becomes a primary concern for afflicted individuals. A number of studies have identified nuclear factor (erythroid-derived 2)-like 2 (Nrf2) as a key regulator in the adaptive survival response to oxidative stress. Elevated expression of Nrf2, combined with heme oxygenase 1 (HO-1) resistance, in the central nervous system is known to elicit key internal and external oxidation protection. Gua Lou Gui Zhi decoction (GLGZD) is a popular traditional Chinese formula with a long history of clinical use in China for the treatment of muscular spasticity following a stroke, epilepsy or a spinal cord injury. However, the mechanism underlying the efficacy of the medicine remains unclear. In the present study, the antioxidative effects of GLGZD were evaluated and the underlying molecular mechanisms were investigated, using hydrogen peroxide (H2O2)-induced rat pheochromocytoma cells (PC12 cells) as an in vitro oxidative stress model of neural cells. Upon application of different concentrations of GLGZD, a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay and ATP measurement were conducted to assess the impact on PC12 cell proliferation. In addition, inverted microscopy observations, and the MTT and ATP assessments, revealed that GLGZD attenuated H2O2-induced oxidative damage and signaling repression in PC12 cells. Furthermore, the mRNA and protein expression levels of Nrf2 and HO-1, which are associated with oxidative stress, were analyzed using reverse transcription quantitative polymerase chain reaction (PCR) and confocal microscopy. Confocal microscopy observations, as well as the quantitative PCR assay, revealed that GLGZD exerted a neuroprotective function against H2O2-induced oxidative damage in PC12 cells. Therefore, the results demonstrated that GLGZD protected PC12 cells injured by H2O2, which may be

  8. Components of Goutengsan in Rat Plasma by Microdialysis Sampling and Its Protection on Aβ1–42-Induced PC12 Cells Injury

    PubMed Central

    Huang, Hou-Cai; Gu, Jun-Fei; Chen, Juan; Hou, Xue-Feng; Zhong, Rong-Ling; Xia, Zhi; Zhao, Di; Yang, Nan; Wang, Jing; Tan, Xiao-Bin; Di, Liu-Qing; Dong, Zi-Bo

    2017-01-01

    Goutengsan, a Chinese herbal formula, potential protection on Alzheimer's disease (AD) has been less reported. In current study, we investigated the protection of Goutengsan on Aβ1–42-induced pheochromocytoma-derived cells (PC12). Furthermore, the components from Goutengsan in rat plasma were identified by microdialysis (MD) for in vivo sampling. Meanwhile, the protection of components identified was also verified. At last, we found that Goutengsan has a potential protective effect on Aβ1–42-induced PC12 cells via reducing cells damage and increasing cells vitality as well as six components (pachymic acid, liquiritin, rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine) which may be effective components. This study helps to understand the treatment of Goutengsan for AD and would facilitate the clinical and further studies for this formula. PMID:28348625

  9. Effects of (-)-sesamin on 6-hydroxydopamine-induced neurotoxicity in PC12 cells and dopaminergic neuronal cells of Parkinson's disease rat models.

    PubMed

    Park, Hyun Jin; Zhao, Ting Ting; Lee, Kyung Sook; Lee, Seung Ho; Shin, Keon Sung; Park, Keun Hong; Choi, Hyun Sook; Lee, Myung Koo

    2015-01-01

    The present study investigated the effects of (-)-sesamin on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity using PC12 cells and dopaminergic neuronal cells of 6-OHDA-lesioned rat model of Parkinson's disease (PD). In PC12 cells, treatment with (-)-sesamin (25 µM) reduced 6-OHDA (100 µM)-induced cell death and induced transient extracellular signal-regulated kinase (ERK1/2) phosphorylation and Bad phosphorylation at Ser112 (BadSer112). In contrast, sustained ERK1/2 phosphorylation, p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK1/2) phosphorylation, and cleaved-caspase-3 activity, all of which were induced by 6-OHDA (100 µM), were inhibited by treatment with (-)-sesamin (25 µM). Furthermore, co-treatment with (-)-sesamin (30 mg/kg, p.o.) once a day for 28 days significantly increased the number of tyrosine hydroxylase-immunopositive neuronal cells and the levels of dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid in the substantia nigra-striatum of 6-OHDA-lesioned rat model of PD with or without L-DOPA treatment. These results suggest that (-)-sesamin protects 6-OHDA-induced cytotoxicity via the activation of transient ERK1/2-BadSer112 system and the inhibition of sustained ERK-p38MAPK-JNK1/2-caspase-3 system in PC12 cells. (-)-Sesamin also shows protective effects on long-term L-DOPA therapy in dopaminergic neuronal cells of PD rat models. (-)-Sesamin may serve as adjuvant therapeutics in PD.

  10. Prevention of cell death by the zinc ion chelating agent TPEN in cultured PC12 cells exposed to Oxygen-Glucose Deprivation (OGD).

    PubMed

    Liu, Zhao; Huang, Yue-yang; Wang, Yu-xiang; Wang, Hong-gang; Deng, Fei; Heng, Bin; Xie, Lai-hua; Liu, Yan-qiang

    2015-01-01

    To elucidate the role of Zn(2+)-associated glutamate signaling pathway and voltage-dependent outward potassium ion currents in neuronal death induced by hypoxia-ischemia, PC12 cells were exposed to Oxygen-Glucose Deprivation (OGD) solution mimicking the hypoxic-ischemic condition in neuron, and the effect of N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn(2+) chelating agent on OGD-induced neuronal death was assessed in the present study. The cell survival rate, apoptosis status, potassium channel currents, intracellular free glutamate concentration and GluR2 expression in PC12 cells exposed to OGD in the absence or presence of TPEN for different time were investigated. The results showed that OGD exposure increased apoptosis, reduced the cell viability (P < 0.01 at 3h, 6h and 24h, respectively compared to control), changed the voltage-dependent outward potassium ion current (increase at 1h, but decrease at 3h) and decreased the concentration of intracellular glutamate (P < 0.05 at 3h and 6h, P < 0.01 at 24h respectively compared to control) and GluR2 expression (P < 0.05 at 3h, 6h and 24h, respectively compared to control) in PC12 cells. TPEN partially reversed the influence resulted from OGD. These results suggest that OGD-induced cell apoptosis and/or death is mediated by the alteration in glutamate signaling pathway and the voltage-dependent outward potassium ion currents, while TPEN effectively prevent cell apoptosis and/or death under hypoxic-ischemic condition.

  11. Yokukansan, a Kampo Medicine, Protects PC12 Cells from Glutamate-Induced Death by Augmenting Gene Expression of Cystine/Glutamate Antiporter System Xc−

    PubMed Central

    Kanno, Hitomi; Kawakami, Zenji; Mizoguchi, Kazushige; Ikarashi, Yasushi; Kase, Yoshio

    2014-01-01

    Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc−, which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells). Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc− subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook. PMID:25551766

  12. Methamphetamine, amphetamine, MDMA ('ecstasy'), MDA and mCPP modulate electrical and cholinergic input in PC12 cells.

    PubMed

    Hondebrink, Laura; Meulenbelt, Jan; Rietjens, Saskia J; Meijer, Marieke; Westerink, Remco H S

    2012-03-01

    Reversal of the dopamine (DA) membrane transporter is the main mechanism through which many drugs of abuse increase DA levels. However, drug-induced modulation of exocytotic DA release by electrical (depolarization) and neurochemical inputs (e.g., acetylcholine (ACh)) may also contribute. We therefore investigated effects of methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and meta-chlorophenylpiperazine (mCPP) (1-1000 μM) on these inputs by measuring drug-induced changes in basal, depolarization- and ACh-evoked intracellular calcium concentrations ([Ca(2+)](i)) using a dopaminergic model (PC12 cells) and Fura 2 calcium imaging. The strongest drug-induced effects were observed on cholinergic input. At 0.1mM all drugs inhibited the ACh-evoked [Ca(2+)](i) increases by 40-75%, whereas ACh-evoked [Ca(2+)](i) increases were nearly abolished following higher drug exposure (1mM, 80-97% inhibition). Additionally, high MDMA and mCPP concentrations increased basal [Ca(2+)](i), but only following prior stimulation with ACh. Interestingly, low concentrations of methamphetamine or amphetamine (10 μM) potentiated ACh-evoked [Ca(2+)](i) increases. Depolarization-evoked [Ca(2+)](i) increases were also inhibited following exposure to high drug concentrations, although drugs were less potent on this endpoint. Our data demonstrate that at high drug concentrations all tested drugs reduce stimulation-evoked increases in [Ca(2+)](i), thereby probably reducing dopaminergic output through inhibition of electrical and cholinergic input. Furthermore, the increases in basal [Ca(2+)](i) at high concentrations of MDMA and mCPP likely increases dopaminergic output. Similarly, the increases in ACh-evoked [Ca(2+)](i) upon cholinergic stimulation following exposure to low concentrations of amphetamines can contribute to drug-induced increases in DA levels observed in vivo. Finally, this study shows that mCPP, which is regularly found in

  13. Nerve growth factor determines survival and death of PC12 cells by regulation of the bcl-x, bax, and caspase-3 genes.

    PubMed

    Rong, P; Bennie, A M; Epa, W R; Barrett, G L

    1999-06-01

    We investigated the effects of nerve growth factor (NGF) and NGF withdrawal on expression of members of the bcl-2 family of genes and caspase-3 in PC12 cells. NGF regulated several members of the bcl-2 family and caspase-3 in a manner consistent with its effect on apoptosis in PC12 cells. Levels of bcl-xl, bcl-xs, and caspase-3 mRNAs were increased by NGF treatment. The increases in caspase-3 and bcl-xs levels should have disposed the cells toward apoptosis but were opposed by the simultaneous increase in bcl-xl level. NGF withdrawal resulted in abrupt down-regulation of bcl-xl and up-regulation of bax, favoring apoptosis. Forced expression of bcl-xl after NGF withdrawal was sufficient to prevent cell death. Cell death was rapid when NGF was withdrawn after 5 days of treatment but relatively slow when NGF was withdrawn after only 1 or 2 days of treatment. This was consistent with the reduced accumulation of caspase-3 mRNA with shorter NGF treatments. These results indicate that Bcl-xl, Bcl-xs, Bax, and caspase-3 are important regulators of apoptosis in PC12 cells. Furthermore, regulation of their mRNA levels is implicated in the signal transduction of NGF.

  14. Protection afforded by quercetin against H2O2-induced apoptosis on PC12 cells via activating PI3K/Akt signal pathway.

    PubMed

    Chen, Liang; Sun, Lejin; Liu, Zhene; Wang, Hongxia; Xu, Cunli

    2016-01-01

    Cell damage and apoptosis induced by oxidative stress have been involved in various neurodegenerative diseases. This study aims to explore the neuro-protective effects of quercetin on PC12 cells apoptosis induced by hydrogen peroxide (H(2)O(2)) and the underlying mechanisms. The cell viability was detected, as well as the production of reactive oxygen species (ROS), lactate dehydrogenase (LDH) leakage, and the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) of the cells in control, H(2)O(2) and quercetin groups. It finally turned out that quercetin might protect PC12 cells against the negative effect of H(2)O(2) by decreasing of LDH release, ROS concentration and MDA level and regaining the GSH-Px and SOD activities. To investigate the mechanism, LY294002 was introduced, the phosphatidylinositol-3-kinase (PI3K) inhibitor. Bax/Bcl-2 ratio and Akt phosphorylation (p-Akt) were examined by Western blot analysis. The data showed that LY294002 almost had the same effects with H(2)O(2), which was also significantly reversed by quercetin could enhance Bax/Bcl-2 ratio and adjust the p-Akt expression, which indicated quercetin might protect PC12 cells against the negative effect of H(2)O(2) via activating the PI3K/Akt signal pathway.

  15. Antioxidant properties and PC12 cell protective effects of a novel curcumin analogue (2E,6E)-2,6-bis(3,5- dimethoxybenzylidene)cyclohexanone (MCH).

    PubMed

    Ao, Gui-Zhen; Chu, Xiao-Jing; Ji, Yuan-Yuan; Wang, Jian-Wen

    2014-03-05

    The antioxidative properties of a novel curcumin analogue (2E,6E)-2,6-bis(3,5-dimethoxybenzylidene)cyclohexanone (MCH) were assessed by several in vitro models, including superoxide anion, hydroxyl radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and PC12 cell protection from H2O2 damage. MCH displayed superior O2•- quenching abilities compared to curcumin and vitamin C. In vitro stability of MCH was also improved compared with curcumin. Exposure of PC12 cells to 150 µM H2O2 caused a decrease of antioxidant enzyme activities, glutathione (GSH) loss, an increase in malondialdehyde (MDA) level, and leakage of lactate dehydrogenase (LDH), cell apoptosis and reduction in cell viability. Pretreatment of the cells with MCH at 0.63-5.00 µM before H2O2 exposure significantly attenuated those changes in a dose-dependent manner. MCH enhanced cellular expression of transcription factor NF-E2-related factor 2 (Nrf2) at the transcriptional level. Moreover, MCH could mitigate intracellular accumulation of reactive oxygen species (ROS), the loss of mitochondrial membrane potential (MMP), and the increase of cleaved caspase-3 activity induced by H2O2. These results show that MCH protects PC12 cells from H2O2 injury by modulating endogenous antioxidant enzymes, scavenging ROS, activating the Nrf2 cytoprotective pathway and prevention of apoptosis.

  16. A study of the effects of flux density and frequency of pulsed electromagnetic field on neurite outgrowth in PC12 cells.

    PubMed

    Zhang, Yang; Ding, Jun; Duan, Wei

    2006-01-01

    The aim of this study was to investigate the influence of pulsed electromagnetic fields with various flux densities and frequencies on neurite outgrowth in PC12 rat pheochromocytoma cells. We have studied the percentage of neurite-bearing cells, average length of neurites and directivity of neurite outgrowth in PC12 cells cultured for 96 hours in the presence of nerve growth factor (NGF). PC12 cells were exposed to 50 Hz pulsed electromagnetic fields with a flux density of 1.37 mT, 0.19 mT and 0.016 mT respectively. The field was generated through a Helmholtz coil pair housed in one incubator and the control samples were placed in another identical incubator. It was found that exposure to both a relatively high flux density (1.37 mT) and a medium flux density (0.19 mT) inhibited the percentage of neurite-bearing cells and promoted neurite length significantly. Exposure to high flux density (1.37 mT) also resulted in nearly 20% enhancement of neurite directivity along the field direction. However, exposure to low flux density field (0.016 mT) had no detectable effect on neurite outgrowth. We also studied the effect of frequency at the constant flux density of 1.37 mT. In the range from 1 approximately 100 Hz, only 50 and 70 Hz pulse frequencies had significant effects on neurite outgrowth. Our study has shown that neurite outgrowth in PC12 cells is sensitive to flux density and frequency of pulsed electromagnetic field.

  17. Ginsenoside-Rd Promotes Neurite Outgrowth of PC12 Cells through MAPK/ERK- and PI3K/AKT-Dependent Pathways

    PubMed Central

    Wu, Song-Di; Xia, Feng; Lin, Xue-Mei; Duan, Kang-Li; Wang, Fang; Lu, Qing-Li; Cao, Huan; Qian, Yi-Hua; Shi, Ming

    2016-01-01

    Panax ginseng is a famous herbal medicine widely used in Asia. Ginsenosides have been identified as the principle active ingredients for Panax ginseng’s biological activity, among which ginsenoside Rd (Rd) attracts extensive attention for its obvious neuroprotective activities. Here we investigated the effect of Rd on neurite outgrowth, a crucial process associated with neuronal repair. PC12 cells, which respond to nerve growth factor (NGF) and serve as a model for neuronal cells, were treated with different concentrations of Rd, and then their neurite outgrowth was evaluated. Our results showed that 10 μM Rd significantly increased the percentages of long neurite- and branching neurite-bearing cells, compared with respective controls. The length of the longest neurites and the total length of neurites in Rd-treated PC12 cells were much longer than that of respective controls. We also showed that Rd activated ERK1/2 and AKT but not PKC signalings, and inhibition of ERK1/2 by PD98059 or/and AKT by LY294002 effectively attenuated Rd-induced neurite outgrowth. Moreover, Rd upregulated the expression of GAP-43, a neuron-specific protein involved in neurite outgrowth, while PD98059 or/and LY294002 decreased Rd-induced increased GAP-43 expression. Taken together, our results provided the first evidence that Rd may promote the neurite outgrowth of PC12 cells by upregulating GAP-43 expression via ERK- and ARK-dependent signaling pathways. PMID:26840295

  18. Minocycline Promotes Neurite Outgrowth of PC12 Cells Exposed to Oxygen-Glucose Deprivation and Reoxygenation Through Regulation of MLCP/MLC Signaling Pathways.

    PubMed

    Tao, Tao; Feng, Jin-Zhou; Xu, Guang-Hui; Fu, Jie; Li, Xiao-Gang; Qin, Xin-Yue

    2017-04-01

    Minocycline, a semi-synthetic second-generation derivative of tetracycline, has been reported to exert neuroprotective effects both in animal models and in clinic trials of neurological diseases. In the present study, we first investigated the protective effects of minocycline on oxygen-glucose deprivation and reoxygenation-induced impairment of neurite outgrowth and its potential mechanism in the neuronal cell line, PC12 cells. We found that minocycline significantly increased cell viability, promoted neurite outgrowth and enhanced the expression of growth-associated protein-43 (GAP-43) in PC12 cells exposed to oxygen-glucose deprivation/reoxygenation injury. In addition, immunoblots revealed that minocycline reversed the overexpression of phosphorylated myosin light chain (MLC) and the suppression of activated extracellular signal-regulated kinase 1/2 (ERK1/2) caused by oxygen-glucose deprivation/reoxygenation injury. Moreover, the minocycline-induced neurite outgrowth was significantly blocked by Calyculin A (1 nM), an inhibitor of myosin light chain phosphatase (MLCP), but not by an ERK1/2 inhibitor (U0126; 10 μM). These findings suggested that minocycline activated the MLCP/MLC signaling pathway in PC12 cells after oxygen-glucose deprivation/reoxygenation injury, which resulted in the promotion of neurite outgrowth.

  19. Tubuloside B from Cistanche salsa rescues the PC12 neuronal cells from 1-methyl-4-phenylpyridinium ion-induced apoptosis and oxidative stress.

    PubMed

    Sheng, Guoqing; Pu, Xiaoping; Lei, Li; Tu, Pengfei; Li, Changling

    2002-11-01

    The neuroprotective effects of tubuloside B, one of the phenylethanoids isolated from the Chinese herbal medicine Cistanche salsa, on 1-methyl-4-phenylpyridinium ion (MPP +)-induced apoptosis and oxidative stress in PC12 neuronal cells were investigated. PC12 cells treated with MPP + underwent apoptotic death as determined by MTT assay, flow cytometry and DNA agarose gel electrophoresis; intracellular accumulation of reactive oxygen species (ROS) was measured by DCFH-DA staining with laser scanning confocal microscopy (LSCM). Simultaneous treatment with tubuloside B markedly attenuated MPP +-induced cytotoxicity, DNA fragmentation, and intracellular accumulation of ROS. These results strongly indicate that tubuloside B prevents MPP +-induced apoptosis and oxidative stress. Tubuloside B may be applied as an antiparkinsonian agent.

  20. Extraction and isolation of potential anti-stroke compounds from flowers of Pueraria lobata guided by in vitro PC12 cell model.

    PubMed

    Li, Sainan; Li, Senlin; Liu, Chengyu; Liu, Chunming; Zhang, Yuchi

    2017-03-24

    A simple and efficient method based on ultrafiltration liquid chromatography-mass spectrometry (UFLC-MS) was applied to rapidly screen and identify ligands for lactate dehydrogenase (LDH) from the flowers of Pueraria lobata, and the compounds were assessed for anti-stroke activity using a PC12 cell model. Seven major isoflavones, kakkalide, 3'-hydroxy puerarin, puerarin, puerarin xyloside, tectoridin, tectorigenin, and ononin, were identified as potent LDH inhibitors. A continuous online method, which consisted of microwave-assisted extraction and countercurrent chromatography (MAE-CCC), was newly developed for scaled-up production of these compounds with high purity and efficiency. This novel approach, using UFLC-MS coupled with MAE-CCC and a PC12 cell model, provided a powerful tool for screening, extraction, and separation of LDH inhibitors from complex samples, and a useful platform for the large-scale production of functional food and nutraceutical ingredients.

  1. Alpha-ketoglutarate and N-acetyl cysteine protect PC12 cells from cyanide-induced cytotoxicity and altered energy metabolism.

    PubMed

    Satpute, R M; Hariharakrishnan, J; Bhattacharya, R

    2008-01-01

    Cyanide is a rapidly acting neurotoxin that inhibits cellular respiration and energy metabolism leading to histotoxic hypoxia. This results in the dissipation of mitochondrial membrane potential (MMP) accompanied by decreased cellular ATP content which in turn is responsible for increased levels of intracellular calcium ions ([Ca(2+)](i)) and total lactic acid content of the cells. Rat pheochromocytoma (PC12) cells possess much of the biochemical machinery associated with synaptic neurons. In the present study, we evaluated the cytoprotective effects of alpha-ketoglutarate (A-KG) and N-acetylcysteine (NAC) against cyanide-induced cytotoxicity and altered energy metabolism in PC12 cells. Cyanide-antagonism by A-KG is attributed to cyanohydrin formation whereas NAC is known for its antioxidant properties. Data on leakage of intracellular lactate dehydrogenase and mitochondrial function (MTT assay) revealed that simultaneous treatment of A-KG (0.5 mM) and NAC (0.25 mM) significantly prevented the cytotoxicity of cyanide. Also, cellular ATP content was found to improve, followed by restoration of MMP, intracellular calcium [Ca(2+)](i) and lactic acid levels. Treatment with A-KG and NAC also attenuated the levels of peroxides generated by cyanide. The study indicates that combined administration of A-KG and NAC protected the cyanide-challenged PC12 cells by resolving the altered energy metabolism. The results have implications in the development of new treatment regimen for cyanide poisoning.

  2. Overexpression of estrogen receptor beta alleviates the toxic effects of beta-amyloid protein on PC12 cells via non-hormonal ligands☆

    PubMed Central

    Wang, Hui; Si, Lihui; Li, Xiaoxi; Deng, Weiguo; Yang, Haimiao; Yang, Yuyan; Fu, Yan

    2012-01-01

    After binding to the estrogen receptor, estrogen can alleviate the toxic effects of beta-amyloid protein, and thereby exert a therapeutic effect on Alzheimer's disease patients. Estrogen can increase the incidence of breast carcinoma and endometrial cancer in post-menopausal women, so it is not suitable for clinical treatment of Alzheimer's disease. There is recent evidence that the estrogen receptor can exert its neuroprotective effects without estrogen dependence. Real-time quantitative PCR and flow cytometry results showed that, compared with non-transfected PC12 cells, adenovirus-mediated estrogen receptor β gene-transfected PC12 cells exhibited lower expression of tumor necrosis factor α and interleukin 1β under stimulation with beta-amyloid protein and stronger protection from apoptosis. The Akt-specific inhibitor Abi-2 decreased the anti-inflammatory and anti-apoptotic effects of estrogen receptor β gene-transfection. These findings suggest that overexpression of estrogen receptor β can alleviate the toxic effect of beta-amyloid protein on PC12 cells, without estrogen dependence. The Akt pathway is one of the potential means for the anti-inflammatory and anti-apoptotic effects of the estrogen receptor. PMID:25722700

  3. Protective Effects of Fucoidan on Aβ25–35 and d-Gal-Induced Neurotoxicity in PC12 Cells and d-Gal-Induced Cognitive Dysfunction in Mice

    PubMed Central

    Wei, Hengyun; Gao, Zixiang; Zheng, Luping; Zhang, Cuili; Liu, Zundong; Yang, Yazong; Teng, Hongming; Hou, Lin; Yin, Yuling; Zou, Xiangyang

    2017-01-01

    Alzheimer’s disease (AD) is a chronic neurodegenerative disease which contributes to memory loss and cognitive decline in the elderly. Fucoidan, extracted from brown algae, is a complex sulfated polysaccharide and potential bioactive compound. In this study, we investigated whether fucoidan protects PC12 cells from apoptosis induced by a combination of beta-amyloid 25–35 (Aβ25–35) and d-galactose (d-Gal), and improves learning and memory impairment in AD model mice. The results indicated that fucoidan could inhibit the release of cytochrome c from the mitochondria to cytosol and activation of caspases, and increase the expression of apoptosis inhibitor proteins (IAPs), including livin and X-linked IAP (XIAP) in PC12 cells damaged by Aβ25–35 and d-Gal-induction. Fucoidan reversed the decreased activity of acetylcholine (ACh) and choline acetyl transferase (ChAT), as well as the increased activity of acetylcholine esterase (AChE), in AD model mice induced by infusion of d-Gal. Furthermore, fucoidan improved antioxidant activity in vitro and in vivo by activation of superoxide dismutase (SOD) and glutathione (GSH). These results suggested that fucoidan could protect PC12 cells from apoptosis and ameliorate the learning and memory impairment in AD model mice, which appeared to be due to regulating the cholinergic system, reducing oxidative stress, and inhibiting the caspase-dependent apoptosis pathway. PMID:28300775

  4. Potentiation of NGF-induced neurite outgrowth in PC12 cells by papaverine: role played by PLC-γ, IP3 receptors.

    PubMed

    Itoh, Kanako; Ishima, Tamaki; Kehler, Jan; Hashimoto, Kenji

    2011-03-04

    Papaverine, an inhibitor of phosphodiesterase (PDE) 10A, is gaining attention for its potential in the treatment of neuropsychiatric diseases such as schizophrenia. However, the precise mechanisms underlying the putative neuroprotective/neurotrophic actions of papaverine remain unclear. Thus, we investigated the effects of papaverine on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Papaverine potentiated NGF-induced neurite outgrowth in PC12 cells in a concentration-dependent manner. In contrast, the selective PDE10A inhibitor MP-10 had no effect on NGF-induced neurite outgrowth. The potentiation of NGF-induced neurite outgrowth by papaverine was blocked by the PLC-γ inhibitor U73122. Furthermore, papaverine's potentiation of NGF-induced neurite outgrowth was also blocked by the co-administration of inositol 1,4,5-trisphosphate (IP(3)) receptor antagonists (xestospongin C and 2-aminoethoxydiphenyl borate (2-APB)) and by reduced expression of IP(3) receptor gene (i.e., itpr1 and itpr3) by siRNA. Our findings suggest that papaverine could potentiate NGF-induced neurite outgrowth, and that activation of PLC-γ and IP(3) receptors might be involved in the mechanism underlying papaverine's potentiation of neurite outgrowth in PC12 cells.

  5. Protective Effects of Fucoidan on Aβ25-35 and d-Gal-Induced Neurotoxicity in PC12 Cells and d-Gal-Induced Cognitive Dysfunction in Mice.

    PubMed

    Wei, Hengyun; Gao, Zixiang; Zheng, Luping; Zhang, Cuili; Liu, Zundong; Yang, Yazong; Teng, Hongming; Hou, Lin; Yin, Yuling; Zou, Xiangyang

    2017-03-16

    Alzheimer's disease (AD) is a chronic neurodegenerative disease which contributes to memory loss and cognitive decline in the elderly. Fucoidan, extracted from brown algae, is a complex sulfated polysaccharide and potential bioactive compound. In this study, we investigated whether fucoidan protects PC12 cells from apoptosis induced by a combination of beta-amyloid 25-35 (Aβ25-35) and d-galactose (d-Gal), and improves learning and memory impairment in AD model mice. The results indicated that fucoidan could inhibit the release of cytochrome c from the mitochondria to cytosol and activation of caspases, and increase the expression of apoptosis inhibitor proteins (IAPs), including livin and X-linked IAP (XIAP) in PC12 cells damaged by Aβ25-35 and d-Gal-induction. Fucoidan reversed the decreased activity of acetylcholine (ACh) and choline acetyl transferase (ChAT), as well as the increased activity of acetylcholine esterase (AChE), in AD model mice induced by infusion of d-Gal. Furthermore, fucoidan improved antioxidant activity in vitro and in vivo by activation of superoxide dismutase (SOD) and glutathione (GSH). These results suggested that fucoidan could protect PC12 cells from apoptosis and ameliorate the learning and memory impairment in AD model mice, which appeared to be due to regulating the cholinergic system, reducing oxidative stress, and inhibiting the caspase-dependent apoptosis pathway.

  6. A novel nerve growth factor-responsive element in the stromelysin-1 (transin) gene that is necessary and sufficient for gene expression in PC12 cells.

    PubMed

    deSouza, S; Lochner, J; Machida, C M; Matrisian, L M; Ciment, G

    1995-04-21

    Stromelysin-1 (ST-1) is an extracellular matrix metalloproteinase whose expression is transcriptionally regulated by nerve growth factor (NGF) in the PC12 rat pheochromocytoma cell line. In this paper, we define sequences in the proximal ST-1 promoter that contain a novel NGF-responsive element(s). We show that this cis-acting promoter element can bind nuclear proteins from both untreated and NGF-treated PC12 cells in a specific and saturable manner and is sufficient to confer NGF-inducibility to a heterologous promoter. At least a portion of this NGF-responsive element lies within a 12-base pair region between positions -241 and -229 of the ST-1 promoter and bears no sequence homology to other known transcriptional elements. In contrast to what has been reported for fibroblasts, an AP1 site centered around position -68 does not seem to be involved in the growth factor regulation of ST-1 in PC12 cells. These results suggest that the NGF regulation of ST-1 gene expression involves different promoter elements, and possibly different transcription factors, from that described for ST-1 induction by other growth factors.

  7. IMM-H004, a novel courmarin derivative, protects against oxygen-and glucose-deprivation/restoration-induced apoptosis in PC12 cells.

    PubMed

    Ji, Hai-jie; Wang, Dong-mei; Hu, Jin-feng; Sun, Ming-na; Li, Gang; Li, Zhi-peng; Wu, Dong-hui; Liu, Gang; Chen, Nai-hong

    2014-01-15

    7-Hydroxy-5-methoxy-4-methyl-3-(4-methylpiperazin-1-yl)-coumarin (IMM-H004) is a novel coumarin derivative synthesized in our laboratory. The purpose of the current study was to determine the neuroprotective effects of IMM-H004 on PC12 cells and its potential mechanism of action. PC12 cells were subject to oxygen and glucose deprivation (OGD) followed by the restoration of oxygen and glucose (R), which mimics ischemia and reperfusion in vivo. Cell viability was determined by MTT assay. DNA fragmentation was analyzed by DNA ladder. ROS and mitochondrial membrane potential were measured by fluorescent microscope and quantified by Image-Pro Express 6.0 software. ATP was measured by luciferin-luciferase assay. The activation of signal-regulated molecules was assessed by the Western blot analysis. OH formation was determined using the Electron Spin Resonance (ESR) trapping technique in combination with 5, 5-dimethyl-1-pyrroline-N-oxide. OGD/R reduced cell viability and induced cell apoptosis, which were both dose-dependently attenuated by IMM-H004. The accumulation of intracellular reactive oxygen species (ROS) and reduced mitochondrial membrane potential observed in PC12 cells treated with OGD/R, which switch on the mitochondrion-dependent apoptotic pathway, were reversed by IMM-H004. ATP production in OGD/R-treated PC12 cells was elevated by IMM-H004, which suggests that it restored the functions of the mitochondria. OGD/R-induced cytochrome c release from the mitochondria reduced the ratio of apoptotic proteins, Bcl-2/Bax, and induced caspase-3 activation and DNA fragmentation. These changes were significantly inhibited by IMM-H004. IMM-H004 also significantly inhibited OH formation, determined by electron spin resonance, which indicates that it is a potent free-radical scavenger. This study has demonstrated that IMM-H004 protects PC12 cells against OGD/R-induced apoptosis, at least in part, by scavenging excessive ROS and inhibiting the mitochondrion

  8. Shikonin protects dopaminergic cell line PC12 against 6-hydroxydopamine-mediated neurotoxicity via both glutathione-dependent and independent pathways and by inhibiting apoptosis.

    PubMed

    Esmaeilzadeh, Emran; Gardaneh, Mossa; Gharib, Ehsan; Sabouni, Farzaneh

    2013-08-01

    We have investigated the mechanism of shikonin function on protection of dopaminergic neurons against 6-OHDA-induced neurotoxicity. Treatment of rat pheochromocytoma cell line PC12 by serial dilutions of shikonin determined 10 μM of the compound as its optimum concentration for protection saving nearly 70 % of the cells against toxicity. Reverse transcription-PCR analysis of shikonin-treated cells showed threefold increase in mRNA levels of glutathione peroxidase-1 (GPX-1) as a representative component of the intracellular anti-oxidant defense system. To elucidate shikonin-GPX1 relationships and maximize protection, we transduced PC12 cells using recombinant lentivirus vectors that harbored GPX-1 coding sequence. This change upregulated GPX-1 expression, increased peroxidase activity and made neuronal cells resistant to 6-OHDA-mediated toxicity. More importantly, addition of shikonin to GPX1-overexpressing PC12 cells augmented GPX-1 protein content by eightfold leading to fivefold increase of enzymatic activity, 91 % cell survival against neurotoxicity and concomitant increases in intracellular glutathione (GSH) levels. Depletion of intracellular GSH rendered all cell groups highly susceptible to toxicity; however, shikonin was capable of partially saving them. Subsequently, GSH-independent superoxide dismutase mRNA was found upregulated by shikonin. As signs of apoptosis inhibition, the compound upregulated Bcl-2, downregulated Bax, and prevented cell nuclei from undergoing morphological changes typical of apoptosis. Also, a co-staining method demonstrated GPX-1 overexpression significantly increases the percent of live cells that is maximized by shikonin treatment. Our data indicate that shikonin as an antioxidant compound protects dopaminergic neurons against 6-OHDA toxicity and enhances their survival via both glutathione-dependent and direct anti-apoptotic pathways.

  9. Antioxidant and neuroprotector effect of Lepidium meyenii (maca) methanol leaf extract against 6-hydroxy dopamine (6-OHDA)-induced toxicity in PC12 cells.

    PubMed

    Rodríguez-Huamán, Ángel; Casimiro-Gonzales, Sandra; Chávez-Pérez, Jorge Antonio; Gonzales-Arimborgo, Carla; Cisneros-Fernández, Richard; Aguilar-Mendoza, Luis Ángel; Gonzales, Gustavo F

    2017-01-08

    Reactive oxygen species (ROS) are normally produced during cell metabolism, there is strong evidence to suggest that ROS produced in excess impair the cell and may be etiologically related to various neurodegenerative diseases. This study was undertaken to examine the effects of Lepidium meyenii (MACA) methanol leaf extract on neurotoxicity in PC12 cell exposed to 6-hydroxydopamine (6-OHDA). Fresh samples of "maca" leaves were processed in order to obtain foliar extracts and to evaluate the neurobiological activity on PC12 cells, subjected to the cytotoxic effect of 6-OHDA through the determination of the capacity antioxidant, cell viability and cytotoxicity assays on PC12 cells. The results of the tests of antioxidant activity, showed maximum values of 2262.37 and 1305.36 expressed in Trolox equivalents (TEAC), for the methanolic and aqueous fractions respectively. Cell viability assays at a dose of 10 μg extract showed an increase of 31% and 60% at 6 and 12 h of pretreatment, respectively. Cytotoxicity assays at the same dose and exposure time showed a 31.4% and 47.8% reduction in lactate dehydrogenase (LDH) activity and an increase in superoxide dismutase (SOD) activity. The results allow us to affirm that the methanolic foliar extract of "maca" presents in vitro neurobiological activity of antioxidant protection, increase in cell viability and reduction of cytotoxicity against oxidative stress generated by 6-OHDA. In conclusion, the present study shows a protective role for Lepidium meyenii leaf extract on 6-OHDA-induced toxicity by an antioxidant effect.

  10. Neuroprotective effect of paeoniflorin on H2O2-induced apoptosis in PC12 cells by modulation of reactive oxygen species and the inflammatory response

    PubMed Central

    LI, PENG; LI, ZHAOHUI

    2015-01-01

    Paeoniflorin (PF) is a product derived from Paeoniae Radix and is commonly prescribed in traditional Chinese medicine. PF has been reported to exhibit neuroprotective, anti-ischemic, antioxidant, anti-inflammatory and anticancer effects. The neuroprotective properties of PF have been demonstrated in animal models of various neuropathologies. The present study investigated the effects of PF on hydrogen peroxide (H2O2)-induced apoptosis in PC12 cells, to improve the understanding of the mechanisms underlying its neuroprotective properties. The H2O2-induced apoptosis of PC12 cells resulted in a reduction in the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein ratio and the activation of caspase-3. PF treatment was observed to reverse the apoptotic process and to modulate the expression levels of a number of apoptosis-associated proteins. Furthermore, PF significantly mitigated the H2O2-induced reduction in cell viability, in addition to scavenging reactive oxygen species and preventing the release of lactate dehydrogenase from the PC12 cells. In addition, the apoptosis-associated activation of nuclear factor (NF)-κB was inhibited in the PF-treated cells, and the expression levels of tumor necrosis factor α and interleukin (IL)-1β were reduced. In conclusion, the present study demonstrated that PF was able to reduce H2O2-induced toxicity by blocking the activation of the neuroinflammatory factor NF-κB. These results suggest that PF may be a valuable neuroprotective agent for the treatment of neurological disease and injury. PMID:26136891

  11. Phytoestrogens genistein and daidzin enhance the acetylcholinesterase activity of the rat pheochromocytoma cell line PC12 by binding to the estrogen receptor.

    PubMed

    Isoda, Hiroko; Talorete, Terence P N; Kimura, Momoko; Maekawa, Takaaki; Inamori, Yuhei; Nakajima, Nobuyoshi; Seki, Humitake

    2002-11-01

    Some compounds derived from plants have been known to possess estrogenic properties and can thus alter the physiology of higher organisms. Genistein and daidzin are examples of these phytoestrogens, which have recently been the subject of extensive research. In this study, genistein and daidzin were found to enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 at concentrations as low as 0.08 muM by binding to the estrogen receptor (ER). Results have shown that this enhancement was effectively blocked by the known estrogen receptor antagonist tamoxifen, indicating the involvement of the ER in AChE induction. That genistein and daidzin are estrogenic were confirmed in a cell proliferation assay using the human breast cancer cell line MCF7. This proliferation was also blocked by tamoxifen, again indicating the involvement of the ER. On the other hand, incubating the PC12 cells in increasing concentrations of 17 beta-estradiol (E2) did not lead to enhanced AChE activity, even in the presence of genistein or daidzin. This suggests that mere binding of an estrogenic compound to the ER does not necessarily lead to enhanced AChE activity. Moreover, the effect of the phytoestrogens on AChE activity cannot be expressed in the presence of E2 since they either could not compete with the natural ligand in binding to the ER or that E2 down-regulates its own receptor. This study clearly suggests that genistein and daidzin enhance AChE activityin PC12 cells by binding to the ER; however, the actual mechanism of enhancement is not known.

  12. Inhibition of Voltage-Gated Calcium Channels After Subchronic and Repeated Exposure of PC12 Cells to Different Classes of Insecticides.

    PubMed

    Meijer, Marieke; Brandsema, Joske A R; Nieuwenhuis, Desirée; Wijnolts, Fiona M J; Dingemans, Milou M L; Westerink, Remco H S

    2015-10-01

    We previously demonstrated that acute inhibition of voltage-gated calcium channels (VGCCs) is a common mode of action for (sub)micromolar concentrations of chemicals, including insecticides. However, because human exposure to chemicals is usually chronic and repeated, we investigated if selected insecticides from different chemical classes (organochlorines, organophosphates, pyrethroids, carbamates, and neonicotinoids) also disturb calcium homeostasis after subchronic (24 h) exposure and after a subsequent (repeated) acute exposure. Effects on calcium homeostasis were investigated with single-cell fluorescence (Fura-2) imaging of PC12 cells. Cells were depolarized with high-K(+) saline to study effects of subchronic or repeated exposure on VGCC-mediated Ca(2+) influx. The results demonstrate that except for carbaryl and imidacloprid, all selected insecticides inhibited depolarization (K(+))-evoked Ca(2+) influx after subchronic exposure (IC50's: approximately 1-10 µM) in PC12 cells. These inhibitory effects were not or only slowly reversible. Moreover, repeated exposure augmented the inhibition of the K(+)-evoked increase in intracellular calcium concentration induced by subchronic exposure to cypermethrin, chlorpyrifos, chlorpyrifos-oxon, and endosulfan (IC50's: approximately 0.1-4 µM). In rat primary cortical cultures, acute and repeated chlorpyrifos exposure also augmented inhibition of VGCCs compared with subchronic exposure. In conclusion, compared with subchronic exposure, repeated exposure increases the potency of insecticides to inhibit VGCCs. However, the potency of insecticides to inhibit VGCCs upon repeated exposure was comparable with the inhibition previously observed following acute exposure, with the exception of chlorpyrifos. The data suggest that an acute exposure paradigm is sufficient for screening chemicals for effects on VGCCs and that PC12 cells are a sensitive model for detection of effects on VGCCs.

  13. Protective effects of aloe-emodin on scopolamine-induced memory impairment in mice and H₂O₂-induced cytotoxicity in PC12 cells.

    PubMed

    Tao, Li; Xie, Jianmei; Wang, Yuting; Wang, Shi; Wu, Shuangchan; Wang, Qiman; Ding, Hong

    2014-12-01

    Aloe-emodin (AE) is one of the most important active components of Rheum officinale Baill. The present study aimed to investigate that AE could attenuate scopolamine-induced cognitive deficits via inhibiting acetylcholinesterase (AChE) activity and modulating oxidative stress. Kunming (KM) mice were received intraperitoneal injection of scopolamine (2 mg/kg) to induce cognitive impairment. Learning and memory performance were assessed in the Morris water maze (MWM). After behavioral testing, the mice were sacrificed and their hippocampi were removed for biochemical assays (superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), AChE and acetylcholine (ACh)). In vitro, we also performed the AChE activity assay and H₂O₂-induced PC12 cells toxicity assay. After 2 h exposure to 200 μM H₂O₂ in PC12 cells, the cytotoxicity were evaluated by cell viability (MTT), nitric oxide (NO)/lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) production. Our results confirmed that AE showed significant improvement in cognitive deficit in scopolamine-induced amnesia animal model. Besides, it increased SOD, GPx activities and ACh content, while decreased the level of MDA and AChE activity in AE treated mice. In addition, AE was found to inhibit AChE activity (IC₅₀ = 18.37 μg/ml) in a dose-dependent manner. Furthermore, preincubation of PC12 cells with AE could prevent cytotoxicity induced by H₂O₂ and reduce significantly extracellular release of NO, LDH and intracellular accumulation of ROS. The study indicated that AE could have neuroprotective effects against Alzheimer's disease (AD) via inhibiting the activity of AChE and modulating oxidative stress.

  14. A small linear peptide encompassing the NGF N-terminus partly mimics the biological activities of the entire neurotrophin in PC12 cells.

    PubMed

    Travaglia, Alessio; Pietropaolo, Adriana; Di Martino, Rossana; Nicoletti, Vincenzo G; La Mendola, Diego; Calissano, Pietro; Rizzarelli, Enrico

    2015-08-19

    Ever since the discovery of its neurite growth promoting activity in sympathetic and sensory ganglia, nerve growth factor (NGF) became the prototype of the large family of neurotrophins. The use of primary cultures and clonal cell lines has revealed several distinct actions of NGF and other neurotrophins. Among several models of NGF activity, the clonal cell line PC12 is the most widely employed. Thus, in the presence of NGF, through the activation of the transmembrane protein TrkA, these cells undergo a progressive mitotic arrest and start to grow electrically excitable neuritis. A vast number of studies opened intriguing aspects of NGF mechanisms of action, its biological properties, and potential use as therapeutic agents. In this context, identifying and utilizing small portions of NGF is of great interest and involves several human diseases including Alzheimer's disease. Here we report the specific action of the peptide encompassing the 1-14 sequence of the human NGF (NGF(1-14)), identified on the basis of scattered indications present in literature. The biological activity of NGF(1-14) was tested on PC12 cells, and its binding with TrkA was predicted by means of a computational approach. NGF(1-14) does not elicit the neurite outgrowth promoting activity, typical of the whole protein, and it only has a moderate action on PC12 proliferation. However, this peptide exerts, in a dose and time dependent fashion, an effective and specific NGF-like action on some highly conserved and biologically crucial intermediates of its intracellular targets such as Akt and CREB. These findings indicate that not all TrkA pathways must be at all times operative, and open the possibility of testing each of them in relation with specific NGF needs, biological actions, and potential therapeutic use.

  15. Mulberry extracts alleviate aβ 25-35-induced injury and change the gene expression profile in PC12 cells.

    PubMed

    Song, Nan; Yang, Hongpeng; Pang, Wei; Qie, Zhiwei; Lu, Hao; Tan, Long; Li, Haiqiang; Sun, Shoudan; Lian, Fuzhi; Qin, Chuan; Jiang, Yugang

    2014-01-01

    Mulberry, which contained high amounts of anthocyanins, has been used in traditional Chinese medicine. Mulberry fruit extracts (ME) have demonstrated the antioxidant activity and neuroprotection. The study was to investigate the neuroprotective efficacy of ME against β-amyloid 25-35- (Aβ 25-35-) induced PC12 cells injury. Cells preincubated with or without ME (200 μg/mL) for 24 h were treated with Aβ 25-35 (20 μmol/L) for another 24 h. Cell viability was assessed by MTT, gene expression profiles were examined by cDNA microarrays, and RT-PCR were used to confirm the results of microarray assays. ME pretreatment was found to neutralize the cytotoxicity and prevent Aβ 25-35-induced cells injury. Analyses of gene expression profile revealed that genes involving cell adhesion, peptidase activity, cytokine activity, ion binding activity, and angiogenesis regulation were significantly modulated by ME pretreatment. Among those genes, Apaf1, Bace2, and Plcb4 were enriched in the "Alzheimer's disease-reference pathway" and downregulated after ME intervention. RT-PCR results showed that ME preincubation could significantly inhibit Aβ 25-35 increased mRNA levels of these three genes. Overall, ME pretreatment could substantially alleviate PC12 cells injury and downregulate expression of AD-related genes, such as Apaf1, Bace2, and Plcb4. This study has a great nutrigenomics interest and brings new and important light in the field of AD intervention.

  16. The neurogenic basic helix-loop-helix transcription factor NeuroD6 enhances mitochondrial biogenesis and bioenergetics to confer tolerance of neuronal PC12-NeuroD6 cells to the mitochondrial stressor rotenone

    SciTech Connect

    Baxter, Kristin Kathleen; Uittenbogaard, Martine; Chiaramello, Anne

    2012-10-15

    The fundamental question of how and which neuronal specific transcription factors tailor mitochondrial biogenesis and bioenergetics to the need of developing neuronal cells has remained largely unexplored. In this study, we report that the neurogenic basic helix-loop-helix transcription factor NeuroD6 possesses mitochondrial biogenic properties by amplifying the mitochondrial DNA content and TFAM expression levels, a key regulator for mitochondrial biogenesis. NeuroD6-mediated increase in mitochondrial biogenesis in the neuronal progenitor-like PC12-NEUROD6 cells is concomitant with enhanced mitochondrial bioenergetic functions, including increased expression levels of specific subunits of respiratory complexes of the electron transport chain, elevated mitochondrial membrane potential and ATP levels produced by oxidative phosphorylation. Thus, NeuroD6 augments the bioenergetic capacity of PC12-NEUROD6 cells to generate an energetic reserve, which confers tolerance to the mitochondrial stressor, rotenone. We found that NeuroD6 induces an adaptive bioenergetic response throughout rotenone treatment involving maintenance of the mitochondrial membrane potential and ATP levels in conjunction with preservation of the actin network. In conclusion, our results support the concept that NeuroD6 plays an integrative role in regulating and coordinating the onset of neuronal differentiation with acquisition of adequate mitochondrial mass and energetic capacity to ensure energy demanding events, such as cytoskeletal remodeling, plasmalemmal expansion, and growth cone formation. -- Highlights: Black-Right-Pointing-Pointer NeuroD6 induces mitochondrial biogenesis in neuroprogenitor-like cells. Black-Right-Pointing-Pointer NeuroD6 augments the bioenergetic reserve of the neuronal PC12-NeuroD6 cells. Black-Right-Pointing-Pointer NeuroD6 increases the mitochondrial membrane potential and ATP levels. Black-Right-Pointing-Pointer NeuroD6 confers tolerance to rotenone via an adaptive

  17. Phycocyanobilin promotes PC12 cell survival and modulates immune and inflammatory genes and oxidative stress markers in acute cerebral hypoperfusion in rats

    SciTech Connect

    Marín-Prida, Javier; Riva, Federica; Pentón-Arias, Eduardo

    2013-10-01

    Since the inflammatory response and oxidative stress are involved in the stroke cascade, we evaluated here the effects of Phycocyanobilin (PCB, the C-Phycocyanin linked tetrapyrrole) on PC12 cell survival, the gene expression and the oxidative status of hypoperfused rat brain. After the permanent bilateral common carotid arteries occlusion (BCCAo), the animals were treated with saline or PCB, taking samples 24 h post-surgery. Global gene expression was analyzed with GeneChip Rat Gene ST 1.1 from Affymetrix; the expression of particular genes was assessed by the Fast SYBR Green RT-PCR Master Mix and Bioplex methods; and redox markers (MDA, PP, CAT, SOD) were evaluated spectrophotometrically. The PCB treatment prevented the H{sub 2}O{sub 2} and glutamate induced PC12 cell injury assessed by the MTT assay, and modulated 190 genes (93 up- and 97 down-regulated) associated to several immunological and inflammatory processes in BCCAo rats. Furthermore, PCB positively modulated 19 genes mostly related to a detrimental pro-inflammatory environment and counteracted the oxidative imbalance in the treated BCCAo animals. Our results support the view of an effective influence of PCB on major inflammatory mediators in acute cerebral hypoperfusion. These results suggest that PCB has a potential to be a treatment for ischemic stroke for which further studies are needed. - Highlights: • Phycocyanobilin (PCB) prevents H{sub 2}O{sub 2} and glutamate induced PC12 cell viability loss. • Anterior cortex and striatum are highly vulnerable to cerebral hypoperfusion (CH). • PCB modulates 190 genes associated to inflammation in acute CH. • PCB regulates 19 genes mostly related to a detrimental pro-inflammatory environment. • PCB restores redox and immune balances showing promise as potential stroke therapy.

  18. Structure-Function Analyses of the Small GTPase Rab35 and Its Effector Protein Centaurin-β2/ACAP2 during Neurite Outgrowth of PC12 Cells*

    PubMed Central

    Etoh, Kan; Fukuda, Mitsunori

    2015-01-01

    The small GTPase Rab35 is a molecular switch for membrane trafficking that regulates a variety of cellular events. We previously showed that Rab35 promotes neurite outgrowth of nerve growth factor-stimulated PC12 cells through interaction with centaurin-β2 (also called ACAP2). Centaurin-β2 is the only Rab35-binding protein reported thus far that exclusively recognizes Rab35 and does not recognize any of the other 59 Rabs identified in mammals, but the molecular basis for the exclusive specificity of centaurin-β2 for Rab35 has remained completely unknown. In this study, we performed deletion and mutation analyses and succeeded in identifying the residues of Rab35 and centaurin-β2 that are crucial for formation of a Rab35·centaurin-β2 complex. We found that two threonine residues (Thr-76 and Thr-81) in the switch II region of Rab35 are responsible for binding centaurin-β2 and that the same residues are dispensable for Rab35 recognition by other Rab35-binding proteins. We also determined the minimal Rab35-binding site of centaurin-β2 and identified two asparagine residues (Asn-610 and Asn-691) in the Rab35-binding site as key residues for its specific Rab35 recognition. We further showed by knockdown-rescue approaches that neither a centaurin-β2 binding-deficient Rab35(T76S/T81A) mutant nor a Rab35 binding-deficient centaurin-β2(N610A/N691A) mutant supported neurite outgrowth of PC12 cells, thereby demonstrating the functional significance of the Rab35/centaurin-β2 interaction during neurite outgrowth of PC12 cells. PMID:25694427

  19. Ac-cel, a novel antioxidant, protects against hydrogen peroxide-induced injury in PC12 cells via attenuation of mitochondrial dysfunction.

    PubMed

    Guo, Xianjun; Chen, Yuting; Liu, Qunfang; Wu, Jian; Wang, Luoyi; Tang, Xican; Zhao, Weimin; Zhang, Haiyan

    2013-07-01

    Oxidative stress has been implicated in pathophysiology of many neurodegenerative diseases (ND) and increased oxidative stress is closely associated with mitochondrial dysfunction. As a result, looking for potent antioxidants, especially those targeting mitochondria, has become an attractive strategy in ND therapy. In this study, we explored protective effects and potential mechanism of Ac-cel, a novel compound, against hydrogen peroxide (H(2)O(2))-induced injury in PC12 cells. Pretreatment of PC12 cells with Ac-cel prior to 24 h of H(2)O(2) exposure markedly attenuated cytotoxicity induced by H(2)O(2) as evidenced by morphological changes and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Ac-cel also exhibited potent antiapoptotic effect demonstrated by results of annexin V and PI staining. The above beneficial effects of Ac-cel were accompanied by improved mitochondrial function, reduced caspase-3 cleavage as well as upregulated ratio of Bcl-2/Bax protein expression. Moreover, Ac-cel pretreatment markedly reversed intracellular reactive oxygen species (ROS) accumulation following 30 min of H(2)O(2) exposure in PC12 cells. Further, subcellular investigation indicated that Ac-cel significantly reduced production of mitochondrial ROS in isolated rat cortical mitochondria. Taken together, the present study, for the first time, reports that Ac-cel pretreatment inhibits H(2)O(2)-stimulated early accumulation of intracellular ROS possibly via reducing mitochondrial ROS production directly and leads to subsequent preservation of mitochondrial function. These results indicate that Ac-cel is a potential drug candidate for treatment of oxidative stress-associated ND.

  20. Quercetin and sesamin protect dopaminergic cells from MPP+-induced neuroinflammation in a microglial (N9)-neuronal (PC12) coculture system.

    PubMed

    Bournival, Julie; Plouffe, Marilyn; Renaud, Justine; Provencher, Cindy; Martinoli, Maria-Grazia

    2012-01-01

    A growing body of evidence indicates that the majority of Parkinson's disease (PD) cases are associated with microglia activation with resultant elevation of various inflammatory mediators and neuroinflammation. In this study, we investigated the effects of 2 natural molecules, quercetin and sesamin, on neuroinflammation induced by the Parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP(+)) in a glial-neuronal system. We first established that quercetin and sesamin defend microglial cells against MPP(+)-induced increases in the mRNA or protein levels of 3 pro-inflammatory cytokines (interleukin-6, IL-1β and tumor necrosis factor-alpha), as revealed by real time-quantitative polymerase chain reaction and enzyme-linked immunoabsorbent assay, respectively. Quercetin and sesamin also decrease MPP(+)-induced oxidative stress in microglial cells by reducing inducible nitric oxide synthase protein expression as well as mitochondrial superoxide radicals. We then measured neuronal cell death and apoptosis after MPP(+) activation of microglia, in a microglial (N9)-neuronal (PC12) coculture system. Our results revealed that quercetin and sesamin rescued neuronal PC12 cells from apoptotic death induced by MPP(+) activation of microglial cells. Altogether, our data demonstrate that the phytoestrogen quercetin and the lignan sesamin diminish MPP(+)-evoked microglial activation and suggest that both these molecules may be regarded as potent, natural, anti-inflammatory compounds.

  1. Protective effect of tetrahydroxystilbene glucoside on 6-OHDA-induced apoptosis in PC12 cells through the ROS-NO pathway.

    PubMed

    Tao, Lizhen; Li, Xiaofeng; Zhang, Lingling; Tian, Jiyu; Li, Xiaobing; Sun, Xin; Li, Xuefen; Jiang, Lin; Zhang, Xiaojun; Chen, Jianzong

    2011-01-01

    Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease. The molecule, 2,3,5,4'-tetrahydr- oxystilbene-2-O-β-D-glucoside (TSG), is a potent antioxidant derived from the Chinese herb, Polygonum multiflorum Thunb. In this study, we investigated the protective effect of TSG against 6-hydroxydopamine-induced apoptosis in rat adrenal pheochromocytoma PC12 cells and the possible mechanisms. Our data demonstrated that TSG significantly reversed the 6-hydroxydopamine-induced decrease in cell viability, prevented 6-hydroxydopamine-induced changes in condensed nuclei and decreased the percentage of apoptotic cells in a dose-dependent manner. In addition, TSG slowed the accumulation of intracellular reactive oxygen species and nitric oxide, counteracted the overexpression of inducible nitric oxide syntheses as well as neuronal nitric oxide syntheses, and also reduced the level of protein-bound 3-nitrotyrosine. These results demonstrate that the protective effects of TSG on rat adrenal pheochromocytoma PC12 cells are mediated, at least in part, by the ROS-NO pathway. Our results indicate that TSG may be effective in providing protection against neurodegenerative diseases associated with oxidative stress.

  2. Neuroprotective effect of crude polysaccharide isolated from the fruiting bodies of Morchella importuna against H2O2-induced PC12 cell cytotoxicity by reducing oxidative stress.

    PubMed

    Xiong, Chuan; Li, Qiang; Chen, Cheng; Chen, Zuqin; Huang, Wenli

    2016-10-01

    Oxidative stress played an essential role in neuronal cell injury through several apoptotic mechanisms associated with many neurodegenerative diseases. Reducing oxidative stress through antioxidants might be a possible strategy that could retard the disease's progression. In order to investigate the neuroprotective role of MIP (the crude polysaccharide extracted from the fruiting bodies of Morchella importuna), the antioxidative activity of MIP against the H2O2-induced cytotoxicity and the underlying preventative mechanisms in rat pheochromocytoma (PC12) cells were illustrated. It was shown that MIP could considerably enhance the viability of PC12 cells exposure to H2O2 and increased the activities of antioxidant enzyme like CAT, GSH-Px and SOD. It also reduced the content of malondialdehyde MDA and caspase-3 activation. In addition, MIP inhibited cell apoptosis via down-regulation of the NF-κB pathway and the p38-JNK pathway as well as activating of the extracellular signal-regulated kinase (ERK). Accordingly, MIP can be used as a promising neuroprotective compound for nervous diseases treatment.

  3. Akt attenuates apoptotic death through phosphorylation of H2A under hydrogen peroxide-induced oxidative stress in PC12 cells and hippocampal neurons

    PubMed Central

    Park, Ji Hye; Kim, Chung Kwon; Lee, Sang Bae; Lee, Kyung-Hoon; Cho, Sung-Woo; Ahn, Jee-Yin

    2016-01-01

    Although the essential role of protein kinase B (PKB)/Akt in cell survival signaling has been clearly established, the mechanism by which Akt mediates the cellular response to hydrogen peroxide (H2O2)-induced oxidative stress remains unclear. We demonstrated that Akt attenuated neuronal apoptosis through direct association with histone 2A (H2A) and phosphorylation of H2A at threonine 17. At early time points during H2O2 exposure of PC12 cells and primary hippocampal neurons, when the cells can tolerate the level of DNA damage, Akt was activated and phosphorylated H2A, leading to inhibition of apoptotic death. At later time points, Akt delivered the NAD+-dependent protein deacetylase Sirtuin 2 (Sirt 2) to the vicinity of phosphorylated H2A in response to irreversible DNA damage, thereby inducing H2A deacetylation and subsequently leading to apoptotic death. Ectopically expressed T17A-substituted H2A minimally interacted with Akt and failed to prevent apoptosis under oxidative stress. Thus Akt-mediated H2A phosphorylation has an anti-apoptotic function in conditions of H2O2-induced oxidative stress in neurons and PC12 cells. PMID:26899247

  4. Sinomenine Protects PC12 Neuronal Cells against H2O2-induced Cytotoxicity and Oxidative Stress via a ROS-dependent Up-regulation of Endogenous Antioxidant System.

    PubMed

    Fan, Hua; Shu, Qing; Guan, Xinlei; Zhao, Jiegang; Yan, Junqiang; Li, Xiangming; Liu, Jiangbo; Jia, Zhaohui; Shi, Jian; Li, Juan

    2017-02-03

    Sinomenine (SN), a purified alkaloid from Chinese herb Sinomenium acutum that was used preferentially in the treatment of rheumatoid diseases, has exerted neuroprotective effects and anti-inflammatory properties in many previous studies. Some studies have revealed that the antioxidant property of SN, acting mainly through inhibiting NADPH oxidase activation, was involved in the beneficial effects of SN. However, SN belongs to the family of dextrorotatory morphinan analogues, which may initiate elevation of reactive oxygen species (ROS) levels. Thus in the present report, we conducted studies to examine its impact and mechanism on the resistance of PC12 neuronal cells to oxidative stress. Precondition with SN (0.1-5 μM) for 12 h significantly decreased H2O2-induced cytotoxicity and remarkably alleviated oxidative injury. However, SN exhibited little direct free radical scavenging property in vitro and induced "appropriate" production of ROS in PC12 cell. Interestingly, the SN-triggering ROS production served as a signal to activate the Nrf2 antioxidant system including Nrf2, HO-1, and NQO-1, which was inhibited by the antioxidant trolox. Furthermore, Nrf2 knockdown largely attenuated the beneficial effects of SN precondition on oxidative stress. In conclusion, our findings suggested that SN increased the resistance to oxidative stress in neuronal cells via a ROS-dependent up-regulation of endogenous antioxidant system, and this mechanism may be involved in the neuroprotection of SN.

  5. Strategy to Suppress Oxidative Damage-Induced Neurotoxicity in PC12 Cells by Curcumin: the Role of ROS-Mediated DNA Damage and the MAPK and AKT Pathways.

    PubMed

    Fu, Xiao-yan; Yang, Ming-feng; Cao, Ming-zhi; Li, Da-wei; Yang, Xiao-yi; Sun, Jing-yi; Zhang, Zong-yong; Mao, Lei-lei; Zhang, Shuai; Wang, Feng-ze; Zhang, Feng; Fan, Cun-dong; Sun, Bao-liang

    2016-01-01

    Oxidative damage plays a key role in causation and progression of neurodegenerative diseases. Inhibition of oxidative stress represents one of the most effective ways in treating human neurologic diseases. Herein, we evaluated the protective effect of curcumin on PC12 cells against H2O2-induced neurotoxicity and investigated its underlying mechanism. The results indicated that curcumin pre-treatment significantly suppressed H2O2-induced cytotoxicity, inhibited the loss of mitochondrial membrane potential (Δψm) through regulation of Bcl-2 family expression, and ultimately reversed H2O2-induced apoptotic cell death in PC12 cells. Attenuation of caspase activation, poly(ADP-ribose) polymerase (PARP) cleavage, DNA damage, and accumulation of reactive oxygen species (ROS) all confirmed its protective effects. Moreover, curcumin markedly alleviated the dysregulation of the MAPK and AKT pathways induced by H2O2. Taken together, our findings suggest that the strategy of using curcumin could be a highly effective way in combating oxidative damage-mediated human neurodegenerative diseases.

  6. Cooperative cytotoxic activity of Zn and Cu in bovine serum albumin-conjugated ZnS/CuS nano-composites in PC12 cancer cells

    NASA Astrophysics Data System (ADS)

    Wang, Hua-Jie; Yu, Xue-Hong; Wang, Cai-Feng; Cao, Ying

    2013-11-01

    Series of self-assembled and mono-dispersed bovine serum albumin (BSA)-conjugated ZnS/CuS nano-composites with different Zn/Cu ratios had been successfully synthesized by a combination method of the biomimetic synthesis and ion-exchange strategy under the gentle conditions. High-resolution transmission electron microscopy observation, Fourier transform infrared spectra and zeta potential analysis demonstrated that BSA-conjugated ZnS/CuS nano-composites with well dispersity had the hierarchical structure and BSA was a key factor to control the morphology and surface electro-negativity of final products. The real-time monitoring by atomic absorption spectroscopy and powder X-ray diffraction revealed that the Zn/Cu ratio of nano-composites could be controlled by adjusting the ion-exchange time. In addition, the metabolic and morphological assays indicated that the metabolic proliferation and spread of rat pheochromocytoma (PC12) cells could be inhibited by nano-composites, with the high anti-cancer activity at a low concentration (4 ppm). What were more important, Zn and Cu in nano-composites exhibited a positive cooperativity at inhibiting cancer cell functions. The microscope observation and biochemical marker analysis clearly revealed that the nano-composites-included lipid peroxidation and disintegration of membrane led to the death of PC12 cells. Summarily, the present study substantiated the potential of BSA-conjugated ZnS/CuS nano-composites as anti-cancer drug.

  7. Neuroprotective effects of adenosine isolated from Cordyceps cicadae against oxidative and ER stress damages induced by glutamate in PC12 cells.

    PubMed

    Olatunji, Opeyemi J; Feng, Yan; Olatunji, Oyenike O; Tang, Jian; Ouyang, Zhen; Su, Zhaoliang; Wang, Dujun; Yu, Xiaofeng

    2016-06-01

    Glutamate has been proven to induce oxidative stress through the formation of reactive oxygen species (ROS) and increased calcium overload which results in neuronal injury, development of neurodegenerative diseases and death. Adenosine is one of the bioactive nucleosides found in Cordyceps cicadae and it has displayed several pharmacological activities including neuroprotection. In this study, the protective effects of adenosine from C. cicadae against glutamate-induce oxidative stress in PC12 cells were evaluated. The exposure of PC12 cells to glutamate (5mM) induced the formation of ROS, increased Ca(2+) influx, endoplasmic reticulum (ER) stress and up regulated the expression of pro-apoptotic factor Bax. However, pretreatment with adenosine markedly increased cell viability, decreased the elevated levels of ROS and Ca(2+) induced by glutamate. Furthermore adenosine increased the activities of GSH-Px and SOD, as well as retained mitochondria membrane potential (MMP), increased Bcl-2/Bax ratio, and reduced the expression of ERK, p38, and JNK. Overall, our results suggest that adenosine may be a promising potential therapeutic agent for the prevention and treatment of neurodegenerative disorders.

  8. Distinctive effect on nerve growth factor-induced PC12 cell neurite outgrowth by two unique neolignan enantiomers from Illicium merrillianum

    NASA Astrophysics Data System (ADS)

    Tian, Xinhui; Yue, Rongcai; Zeng, Huawu; Li, Honglin; Shan, Lei; He, Weiwei; Shen, Yunheng; Zhang, Weidong

    2015-11-01

    Merrillianoid (1), a racemic neolignan possessing the characteristic benzo-2,7-dioxabicyclo[3.2.1]octane moiety, was isolated from the branches and leaves of Illicium merrillianum. Chiral separation of 1 gave two enantiomers (+)-1 and (-)-1. The structure of 1 was established by comprehensive spectroscopic analysis and single crystal X-ray diffraction. The absolute configurations of enantiomers were determined by quantum mechanical calculation. Compound (+)-1 exhibited a better neurotrophic activity than racemate 1 by promoting nerve growth factor (NGF) induced PC12 cell neurite outgrowth, while (-)-1 showed a distinctive inhibitory effect. Furthermore, a mechanism study indicated that the two enantiomers influenced NGF-induced neurite outgrowth of PC12 cells possibly by interacting with the trkA receptor, and extracellular signal regulated kinases 1/2 (ERK1/2) and mitogen-activated protein kinase (MEK) in Ras/ERK signal cascade. But the phosphorylation level of serine/threonine kinase Akt1 and Akt2 in PI3K/Akt signal pathway showed no significant difference between (+)-1 and (-)-1.

  9. Identification of a potent and selective σ₁ receptor agonist potentiating NGF-induced neurite outgrowth in PC12 cells.

    PubMed

    Rossi, Daniela; Pedrali, Alice; Urbano, Mariangela; Gaggeri, Raffaella; Serra, Massimo; Fernández, Leyden; Fernández, Michael; Caballero, Julio; Ronsisvalle, Simone; Prezzavento, Orazio; Schepmann, Dirk; Wuensch, Bernhard; Peviani, Marco; Curti, Daniela; Azzolina, Ornella; Collina, Simona

    2011-11-01

    Herein we report the synthesis, drug-likeness evaluation, and in vitro studies of new sigma (σ) ligands based on arylalkenylaminic scaffold. For the most active olefin the corresponding arylalkylamine was studied. Novel arylalkenylamines generally possess high σ(1) receptor affinity (K(i) values <25 nM) and good σ(1)/σ(2) selectivity (K(i)σ(2) >100). Particularly, the piperidine derivative (E)-17 and its arylalkylamine analog (R,S)-33 were observed to be excellent σ(1) receptor ligands (K(i)=0.70 and 0.86 nM, respectively) and to display significantly high selectivity over σ(2), μ-, and κ-opioid receptors and phencyclidine (PCP) binding site of the N-methyl-d-aspartate (NMDA) receptors. Moreover in PC12 cells (R,S)-33 promoted the nerve growth factor (NGF)-induced neurite outgrowth and elongation. Co-administration of the selective σ(1) receptor antagonist BD-1063 totally counteracted this effect, confirming that σ(1) receptors are involved in the (R,S)-33 modulation of the NGF effect in PC12 cells and suggesting a σ(1) agonist profile. As a part of our work, a threedimensional σ(1) pharmacophore model was also developed employing GALAHAD methodology. Only active compounds were used for deriving this model. The model included two hydrophobes and a positive nitrogen as relevant features and it was able to discriminate between molecules with and without affinity toward σ(1) receptor subtype.

  10. Distinctive effect on nerve growth factor-induced PC12 cell neurite outgrowth by two unique neolignan enantiomers from Illicium merrillianum

    PubMed Central

    Tian, Xinhui; Yue, Rongcai; Zeng, Huawu; Li, Honglin; Shan, Lei; He, Weiwei; Shen, Yunheng; Zhang, Weidong

    2015-01-01

    Merrillianoid (1), a racemic neolignan possessing the characteristic benzo-2,7-dioxabicyclo[3.2.1]octane moiety, was isolated from the branches and leaves of Illicium merrillianum. Chiral separation of 1 gave two enantiomers (+)−1 and (−)−1. The structure of 1 was established by comprehensive spectroscopic analysis and single crystal X-ray diffraction. The absolute configurations of enantiomers were determined by quantum mechanical calculation. Compound (+)−1 exhibited a better neurotrophic activity than racemate 1 by promoting nerve growth factor (NGF) induced PC12 cell neurite outgrowth, while (−)−1 showed a distinctive inhibitory effect. Furthermore, a mechanism study indicated that the two enantiomers influenced NGF-induced neurite outgrowth of PC12 cells possibly by interacting with the trkA receptor, and extracellular signal regulated kinases 1/2 (ERK1/2) and mitogen-activated protein kinase (MEK) in Ras/ERK signal cascade. But the phosphorylation level of serine/threonine kinase Akt1 and Akt2 in PI3K/Akt signal pathway showed no significant difference between (+)−1 and (−)−1. PMID:26585042

  11. Inhibition of 6-hydroxydopamine-induced PC12 cell apoptosis by olive (Olea europaea L.) leaf extract is performed by its main component oleuropein.

    PubMed

    Pasban-Aliabadi, Hamzeh; Esmaeili-Mahani, Saeed; Sheibani, Vahid; Abbasnejad, Mehdi; Mehdizadeh, Anahita; Yaghoobi, Mohammad Mehdi

    2013-04-01

    Parkinson disease (PD) is the most common progressive neurodegenerative disorder characterized by progressive death of midbrain dopaminergic neurons. Most neurodegenerative disease treatments are, at present, palliative. However, some natural herbal products have been shown to rescue neurons from death and apoptosis in some of neurodegenerative diseases. Not only Olea europaea L. olive oil, but also the leaves of this plant have been used for medical purposes. Olive leaf extract (OLE) is being used by people as a drink across the world and as an integral ingredient in their desire to maintain and improve their health. Here, we investigated the effects of OLE and its main phenolic component oleuropein on 6-hydroxydopamine (6-OHDA)-induced toxicity in rat adrenal pheochromocytoma (PC12) cells as an in vitro model of PD. Cell damage was induced by 150 μM 6-OHDA. The cell survival rate was examined by MTT assay. Generation of intra-cellular reactive oxygen species (ROS) was studied using fluorescence spectrophotometry. Immunoblotting and DNA analysis were also employed to determine the levels of biochemical markers of apoptosis in the cells. The data showed that 6-OHDA could decrease the viability of the cells. In addition, intra-cellular ROS, activated caspase 3, Bax/Bcl-2 ratio, as well as DNA fragmentation were significantly increased in 6-OHDA-treated cells. Incubation of cells with OLE (400 and 600 μg/mL) and oleuropein (20 and 25 μg/mL) could decrease cell damage and reduce biochemical markers of cell death. The results suggest that OLE and oleuropein have anti-oxidant protective effects against 6-OHDA-induced PC12 cell damage. The protective effects of OLE and oleuropein are correlative with their anti-oxidative and anti-apoptotic properties and suggest their therapeutic potential in the treatment of PD.

  12. Tyrosine hydroxylase is activated and phosphorylated at different sites in rat pheochromocytoma PC 12 cells treated with phorbol ester and forskolin

    SciTech Connect

    Tachikawa, E.; Tank, A.W.; Weiner, D.H.; Mosimann, W.F.; Yanagihara, N.; Weiner, N.

    1986-03-01

    The effects of phorbol ester (4..beta..-phorbol, 12..beta..-myristate, 13..cap alpha..-acetate; TPA), an activator of Ca/sup + +//phospholipid-dependent protein kinase (PK-C), and forskolin, which stimulates adenylate cyclase and cyclic AMP-dependent protein kinase (cAMP-PK), on the activation and phosphorylation of tyrosine hydroxylase (TH) in rat pheochromocytoma (PC 12) cells were examined. Incubation of the cells with TPA (0.01-1 ..mu..M) or forskolin (0.01-0.1 ..mu..M) produces increases in activation and phosphorylation of TH in a concentration-dependent manner. The stimulatory effects of TPA are dependent on extracellular Ca/sup + +/ and are inhibited by pretreatment of the cells with trifluoperazine (TFP). The effects of forskolin are independent of Ca/sup + +/ and are not inhibited by TFP. In cells treated with forskolin, the time course of the increase in cAMP correlates with the increases in TH activity and phosphorylation. cAMP levels do not increase in cells treated with TPA. There is an increase in the phosphorylation of only one tryptic phosphopeptide derived from TH in cells treated with either forskolin or TPA. The peptide phosphorylated in TPA-treated cells exhibits different elution characteristics on HPLC from that in forskolin-treated cells. The authors conclude that TH in PC 12 cells is phosphorylated on different sites by cAMP-PK and PK-C. Phosphorylation of either of these sites is associated with enzyme activation.

  13. Minocycline attenuates both OGD-induced HMGB1 release and HMGB1-induced cell death in ischemic neuronal injury in PC12 cells

    SciTech Connect

    Kikuchi, Kiyoshi; Kawahara, Ko-ichi; Biswas, Kamal Krishna; Ito, Takashi; Tancharoen, Salunya; Morimoto, Yoko; Matsuda, Fumiyo; Oyama, Yoko; Takenouchi, Kazunori; Miura, Naoki; Arimura, Noboru; Nawa, Yuko; Meng, Xiaojie; Shrestha, Binita; Arimura, Shinichiro; and others

    2009-07-24

    High mobility group box-1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic insult and exacerbates brain tissue damage in rats. Minocycline is a semisynthetic second-generation tetracycline antibiotic which has recently been shown to be a promising neuroprotective agent. In this study, we found that minocycline inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated PC12 cells and triggered the activation of p38mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK1/2). The ERK kinase (MEK)1/2 inhibitor U-0126 and p38MAPK inhibitor SB203580 blocked HMGB1 release in response to OGD. Furthermore, HMGB1 triggered cell death in a dose-dependent fashion. Minocycline significantly rescued HMGB1-induced cell death in a dose-dependent manner. In light of recent observations as well as the good safety profile of minocycline in humans, we propose that minocycline might play a potent neuroprotective role through the inhibition of HMGB1-induced neuronal cell death in cerebral infarction.

  14. Enhancing Beta-Catenin Activity via GSK3beta Inhibition Protects PC12 Cells against Rotenone Toxicity through Nurr1 Induction

    PubMed Central

    Wei, Lei; Mo, Mingshu; Feng, Junmin; Sun, Congcong; Xiao, Yousheng; Luo, Qin; Li, Shaomin; Yang, Xinling; Xu, Pingyi

    2016-01-01

    Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic (DA) neurons in the substantial nigra pars compacta. Increasing evidence showed that Wnt/β-catenin pathway and the orphan nuclear receptor Nurr1 play crucial roles in the survival and functional maintenance of DA neurons in the midbrain and GSK-3β antagonists LiCl and SB216763 were used to activate Wnt/β-catenin pathway experimentally. However, the detail mechanism underlying the neuroprotection against apoptosis on DA neuron is still unclear and the interaction between Wnt/β-catenin and Nurr1 remains undisclosed. In this study, using cell biological assay we investigated the function of Wnt/β-catenin and its crosstalk with Nurr1 on the course of PC12 cell degeneration in vitro. Our data showed that PC12 cell viability was inhibited by rotenone, but attenuated by GSK-3β antagonists LiCl or SB216763. The activity of Wnt/β-catenin pathway was deregulated on exposure of rotenone in a concentration-dependent manner. After the interference of β-catenin with siRNA, LiCl or SB216763 failed to protect PC12 cells from apoptosis by the rotenone toxicity. Our data confirmed that Wnt/β-catenin signaling activated by LiCl or SB216763 enhanced Nurr1 expression to 2.75 ± 0.55 and 4.06 ± 0.41 folds respectively compared with control detected by real-time PCR and the interaction of β-catenin with Nurr1 was identified by co-immunoprecipitate analysis. In conclusion, the data suggested that Wnt/β-catenin and Nurr1 are crucial factors in the survival of DA neurons, and the activation of Wnt/β-catenin pathway exerts protective effects on DA neurons partly by mean of a co-active pattern with Nurr1. This finding may shed a light on the potential treatment of Parkinson disease. PMID:27045591

  15. Botulinum toxin type A targets RhoB to inhibit lysophosphatidic acid-stimulated actin reorganization and acetylcholine release in nerve growth factor-treated PC12 cells.

    PubMed

    Ishida, Hiroshi; Zhang, Xieping; Erickson, Kelly; Ray, Prabhati

    2004-09-01

    Botulinum toxin type A (BoNT/A) produced by Clostridium botulinum inhibits Ca2+-dependent acetylcholine (ACh) release (neuroexocytosis) at peripheral neuromuscular junctions, sometimes causing neuromuscular paralysis. This inhibitory effect is attributed to its metalloprotease activity to cleave the 25-kDa synaptosomal-associated protein, which is essential for the exocytotic machinery. However, deletion of this protein does not result in a complete block of neuroexocytosis, suggesting that botulinum-mediated inhibition may occur via another mechanism. Rho GTPases, a class of small GTP-binding proteins (G proteins), control actin cytoskeletal organization, thereby regulating a variety of cellular functions in various cells, including neuronal cells. We have shown that the G protein activator lysophosphatidic acid (LPA) triggered actin reorganization followed by Ca2+-dependent ACh release in nerve growth factor-treated PC12 cells and that BoNT/A blocked both events through degradation of RhoB by the proteasome. Overexpression of wild-type RhoB caused actin reorganization and enhanced the release of ACh by LPA to overcome toxin's inhibitory effect on actin reorganization/exocytosis stimulated by LPA, whereas overexpression of a dominant negative RhoB inhibited ACh release, regardless of LPA and/or toxin treatment. Finally, a knockdown of the RhoB gene via sequence-specific, post-transcriptional gene silencing reduced RhoB expression in PC12 cells, resulting in total inhibition of both actin reorganization and ACh release induced by LPA. We conclude that the RhoB signaling pathway regulates ACh release via actin cytoskeletal reorganization and that botulinum toxin inhibits neuroexocytosis by targeting RhoB pathway.

  16. TRPC6 channel-mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK, PI3K, and CAMKIV signaling.

    PubMed

    Heiser, Jeanine H; Schuwald, Anita M; Sillani, Giacomo; Ye, Lian; Müller, Walter E; Leuner, Kristina

    2013-11-01

    The non-selective cationic transient receptor canonical 6 (TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin-induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient-mediated changes in synaptic plasticity, ranging from calmodulin-mediated Ras-induced signaling cascades comprising the mitogen-activated protein kinase, PI3K signal transduction pathways as well as Ca(2+) /calmodulin-dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6-mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/mitogen-activated protein kinase/extracellular signal-regulated kinases, phosphatidylinositide 3-kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP-response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract-mediated antidepressant activity. Alterations in synaptic plasticity are considered to play an important role in the pathogenesis of depression. Beside several other proteins, TRPC6 channels regulate synaptic plasticity. This study demonstrates that different pathways including Ras/MEK/ERK, PI3K/Akt, and CAMKIV are involved in the improvement of synaptic plasticity by the TRPC6 activator hyperforin, the antidepressant active constituent of St. John

  17. Bioactive profiles, antioxidant activities, nitrite scavenging capacities and protective effects on H2O2-injured PC12 cells of Glycyrrhiza glabra L. leaf and root extracts.

    PubMed

    Dong, Yi; Zhao, Mouming; Zhao, Tiantian; Feng, Mengying; Chen, Huiping; Zhuang, Mingzhu; Lin, Lianzhu

    2014-06-30

    This study compared the total flavonoid content of Glycyrrhiza glabra L. leaf and root extracts. Results suggested that the total flavonoid content in the leaf extract was obviously higher than that in the root extract. Pinocembrin, the main compound in the leaf extract after purification by column chromatography, showed good antioxidant activity and nitrite scavenging capacity, but moderate inhibitory effect on mushroom tyrosinase. Liquiritin was the main compound in root extract and possessed strong inhibitory effect on mushroom tyrosinase. Both compounds exhibited significant protection effect on H2O2-injured PC12 cells at a low concentration. These results indicate that Glycyrrhiza glabra L. leaf is potential as an important raw material for functional food.

  18. The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells.

    PubMed

    Mobarak, C D; Anderson, K D; Morin, M; Beckel-Mitchener, A; Rogers, S L; Furneaux, H; King, P; Perrone-Bizzozero, N I

    2000-09-01

    The RNA-binding protein HuD binds to a regulatory element in the 3' untranslated region (3' UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels were controlled by transfection with either antisense (pDuH) or sense (pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP-43 protein and mRNA, and these levels remained low even after nerve growth factor (NGF) stimulation, a treatment that is normally associated with protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. The GAP-43 mRNA was stabilized in these cells, leading to an increase in the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to grow short spontaneous neurites, a process that required the presence of GAP-43. In conclusion, our results suggest that HuD plays a critical role in PKC-mediated neurite outgrowth in PC12 cells and that this protein does so primarily by promoting the stabilization of the GAP-43 mRNA.

  19. Conformational states of syntaxin-1 govern the necessity of N-peptide binding in exocytosis of PC12 cells and Caenorhabditis elegans

    PubMed Central

    Park, Seungmee; Bin, Na-Ryum; Michael Rajah, Maaran; Kim, Byungjin; Chou, Ting-Chieh; Kang, Soo-young Ann; Sugita, Kyoko; Parsaud, Leon; Smith, Matthew; Monnier, Philippe P.; Ikura, Mitsuhiko; Zhen, Mei; Sugita, Shuzo

    2016-01-01

    Syntaxin-1 is the central SNARE protein for neuronal exocytosis. It interacts with Munc18-1 through its cytoplasmic domains, including the N-terminal peptide (N-peptide). Here we examine the role of the N-peptide binding in two conformational states (“closed” vs. “open”) of syntaxin-1 using PC12 cells and Caenorhabditis elegans. We show that expression of “closed” syntaxin-1A carrying N-terminal single point mutations (D3R, L8A) that perturb interaction with the hydrophobic pocket of Munc18-1 rescues impaired secretion in syntaxin-1–depleted PC12 cells and the lethality and lethargy of unc-64 (C. elegans orthologue of syntaxin-1)-null mutants. Conversely, expression of the “open” syntaxin-1A harboring the same mutations fails to rescue the impairments. Biochemically, the L8A mutation alone slightly weakens the binding between “closed” syntaxin-1A and Munc18-1, whereas the same mutation in the “open” syntaxin-1A disrupts it. Our results reveal a striking interplay between the syntaxin-1 N-peptide and the conformational state of the protein. We propose that the N-peptide plays a critical role in intracellular trafficking of syntaxin-1, which is dependent on the conformational state of this protein. Surprisingly, however, the N-peptide binding mode seems dispensable for SNARE-mediated exocytosis per se, as long as the protein is trafficked to the plasma membrane. PMID:26700321

  20. The protective effect of eicosapentaenoic acid-enriched phospholipids from sea cucumber Cucumaria frondosa on oxidative stress in PC12 cells and SAMP8 mice.

    PubMed

    Wu, Feng-Juan; Xue, Yong; Liu, Xiao-Fang; Xue, Chang-Hu; Wang, Jing-Feng; Du, Lei; Takahashi, Koretaro; Wang, Yu-Ming

    2014-01-01

    Alzheimer's disease (AD) is a common neurodegenerative disorders, in which oxidative stress plays an important role. The present study investigated the effect of eicosapentaenoic acid-enriched phospholipids (EPA-enriched PL) from the sea cucumber Cucumaria frondosa on oxidative injury in PC12 cells induced by hydrogen peroxide (H2O2) and tert-butylhydroperoxide (t-BHP). We also studied the effect of EPA-enriched PL on learning and memory functions in senescence-accelerated prone mouse strain 8 (SAMP8) in vivo. Pretreatment with EPA-enriched PL resulted in an enhancement of survival in a dose-dependent manner in H2O2 or t-BHP damaged PC12 cells. EPA-enriched PL pretreatment could also reduce the leakage of lactate dehydrogenase (LDH), and increase the intracellular total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity compared with the H2O2 or t-BHP group. The down-regulated Bcl-2 mRNA level and up-regulated Bax, Caspase-9, and Caspase-3 mRNA expression induced by H2O2 or t-BHP could be restored by EPA-enriched PL pretreatment. These results demonstrated that EPA-enriched PL exhibited its neuroprotective effects by virtue of its antioxidant activity, which might be achieved by inhibiting the mitochondria-dependent apoptotic pathway. The neuroprotective effect of EPA-enriched PL was also verified in vivo test: the EPA-enriched PL administration prevented the development of learning and memory impairments in SAMP8 mice. Our results indicated that EPA-enriched PL could offer an efficient and novel strategy to explore novel drugs or functional food for neuronprotection and cognitive improvement.

  1. Luteolin and Apigenin Attenuate 4-Hydroxy-2-Nonenal-Mediated Cell Death through Modulation of UPR, Nrf2-ARE and MAPK Pathways in PC12 Cells.

    PubMed

    Wu, Pei-Shan; Yen, Jui-Hung; Kou, Mei-Chun; Wu, Ming-Jiuan

    2015-01-01

    Luteolin and apigenin are dietary flavones and exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) has been implicated as a causative agent in the development of neurodegenerative disorders. This study investigates the cytoprotective effects of luteolin and apigenin against 4-HNE-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Both flavones restored cell viability and repressed caspase-3 and PARP-1 activation in 4-HNE-treated cells. Luteolin also mitigated 4-HNE-mediated LC3 conversion and reactive oxygen species (ROS) production. Luteolin and apigenin up-regulated 4-HNE-mediated unfolded protein response (UPR), leading to an increase in endoplasmic reticulum chaperone GRP78 and decrease in the expression of UPR-targeted pro-apoptotic genes. They also induced the expression of Nrf2-targeted HO-1 and xCT in the absence of 4-HNE, but counteracted their expression in the presence of 4-HNE. Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin. Consistently, enhanced phosphorylation of JNK and p38 MAPK was observed in luteolin- and apigenin-treated cells. In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity. Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity.

  2. Surface Plasmon Resonance for Measuring Exocytosis from Populations of PC12 Cells: Mechanisms of Signal Formation and Assessment of Analytical Capabilities.

    PubMed

    Moreira, Beatriz; Tuoriniemi, Jani; Kouchak Pour, Naghmeh; Mihalčíková, Lýdia; Safina, Gulnara

    2017-03-07

    Because of cell to cell variation, it is difficult to obtain statistically significant data on the frequency of exocytosis events (Rexocytosis, t(-1) m(-2)) with traditional single cell electrophysiological or fluorescence microscopy based methods. Here we take the first steps toward a rapid cost-effective surface plasmon resonance (SPR) based method for measuring the Rexocytosis for populations of PC12 cells. The conditions for culturing confluent monolayers on the sensor slides were optimized, and neurotransmitter exocytosis was evoked by injecting solutions with elevated [K(+)]. Exocytosis caused a shift of the resonance angle (Δθr) that was linearly proportional to Rexocytosis. The Δθr was mainly due to elevated concentration of secretory vesicles close to the cell membrane. The increased vesicle concentration thus acted as a proxy for the Rexocytosis that could not be measured directly. The Δθr was calibrated for Rexocytosis using single cell amperometry on parallel cell cultures. The cell populations were large enough for variation in responses between sensor slides to only reflect actual differences in biological condition. The applicability for drug screening is demonstrated by studying the effects of EGTA, reserpine, and prolonged stimulation by K(+).

  3. Consecutive Gated Injection-Based Microchip Electrophoresis for Simultaneous Quantitation of Superoxide Anion and Nitric Oxide in Single PC-12 Cells.

    PubMed

    Li, Lu; Li, Qingling; Chen, Peilin; Li, Zhongyi; Chen, Zhenzhen; Tang, Bo

    2016-01-05

    As important reactive oxygen species (ROS) and reactive nitrogen species (RNS), cellular superoxide anion (O2(•-)) and nitric oxide (NO) play significant roles in numerous physiological and pathological processes. Cellular O2(•-) and NO also have a close relationship and always interact with each other. Thus, the simultaneous detection of intracellular O2(•-) and NO, especially at the single-cell level, is important. In this paper, we present a novel method to simultaneously detect and quantify O2(•-) and NO in single cells using microchip electrophoresis based on a new consecutive gated injection method. This novel injection method achieved consecutive manipulation of single cells, guaranteeing an almost constant volumetric flow rate and thus good quantitative reproducibility. After cellular content separation by microchip electrophoresis and detection by laser-induced fluorescence (MCE-LIF), O2(•-) and NO in single PC-12 cells were simultaneously quantified in an automated fashion. This is the first report of consecutive absolute quantitation at the single-cell level. The quantitative results obtained from single cells is beneficial for deep understanding of the biological roles of cellular O2(•-) and NO. This new method constitutes a consecutive, accurate way to study the synergistic function of O2(•-) and NO and other biomolecules in various biological events at the single-cell level.

  4. Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening

    EPA Science Inventory

    Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process un...

  5. A combination of four effective components derived from Sheng-mai san attenuates hydrogen peroxide-induced injury in PC12 cells through inhibiting Akt and MAPK signaling pathways.

    PubMed

    Cao, Guo-Sheng; Li, Shao-Xia; Wang, Yan; Xu, Ying-Qiong; Lv, Yan-Ni; Kou, Jun-Ping; Yu, Bo-Yang

    2016-07-01

    The present study was designed to investigate whether a combination of four effective components derived from Sheng-mai san (SMXZF; ginsenoside Rb1: ginsenoside Rg1: DT-13: Schizandrol A as 6 : 9 : 4 : 5) could attenuate hydrogen peroxide (H2O2)-induced injury in PC12 cells, focusing on the Akt and MAPK pathways . The PC12 cells were exposed to H2O2 (400 μmol·L(-1)) for 1 h in the presence or absence of SMXZF pre-treatment for 24 h. Cell viability was measured by MTT assay. The efflux of lactate dehydrogenase (LDH), the intracellular content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD), and caspase-3 were also determined. Cell apoptosis was measured by Hoechst 33342 staining and Annexin V-FITC/PI staining method. The expression of Bcl-2, Bax, cleaved caspase-3, Akt, and MAPKs were detected by Western blotting analyses. SMXZF pretreatment significantly increased the cell viability and SOD activity and improved the cell morphological changes, while reduced the levels of LDH and MDA at the concentrations of 0.1, 1 and 10 μg·mL(-1). SMXZF also inhibited H2O2-induced apoptosis in PC12 cells. Moreover, SMXZF reduced the activity of caspase-3, up-regulated the protein ratio of Bcl-2 and Bax and inhibited the expression of cleaved caspase-3, p-Akt, p-p38, p-JNK and p-ERK1/2 in H2O2-induced PC12 cells. Co-incubation of Akt inhibitor or p38 inhibitor partly attenuated the protection of SMXZF against H2O2-injured PC12 cells. In conclusion, our findings suggested that SMXZF attenuated H2O2-induced injury in PC12 cells by inhibiting Akt and MAPKs signaling pathways, which might shed insights on its neuroprotective mechanism.

  6. 20C, a bibenzyl compound isolated from Gastrodia elata, protects PC12 cells against rotenone-induced apoptosis via activation of the Nrf2/ARE/HO-1 signaling pathway

    PubMed Central

    Huang, Ju-yang; Yuan, Yu-he; Yan, Jia-qing; Wang, Ya-nan; Chu, Shi-feng; Zhu, Cheng-gen; Guo, Qing-lan; Shi, Jian-gong; Chen, Nai-hong

    2016-01-01

    Aim: Our preliminary study shows that a bibenzyl compound isolated from Gastrodia elata, 2-[4-hydroxy-3-(4-hydroxybenzyl)benzyl]-4-(4-hydroxybenzyl)phenol (designated 20C), protects PC12 cells against H2O2-induced injury. In this study we investigated whether 20C exerted neuroprotective action in a cell model of Parkinson's disease. Methods: A cell model of Parkinson's disease was established in PC12 cells by exposure to rotenone (4 μmol/L) for 48 h. Cell viability and apoptosis were assessed, and intracellular ROS level and the mitochondrial membrane potential (MMP) were detected. The expression of apoptosis-related proteins Bax, Bcl-2, cytochrome c, cleaved caspase-3, and oxidative stress-related proteins Nrf2, HO-1 and NQO1 were examined using Western blotting. The mRNA levels of HO-1 and NQO1 were determined with RT-PCR. The nuclear translocation of Nrf2 was observed with immunofluorescence staining. Results: Treatment with rotenone significantly increased the number of apoptotic cells, accompanied by marked increases in the Bax/Bcl-2 ratio, cytochrome c release and caspase-3 activation. Rotenone also increased ROS accumulation, reduced MMP, and increased the nuclear translocation of Nrf2 as well as the mRNA and protein levels of the Nrf2 downstream target genes HO-1 and NQO1 in PC12 cells. Co-treatment with 20C (0.01–1 μmol/L) dose-dependently attenuated rotenone-induced apoptosis and oxidative stress in PC12 cells. Nrf2 knockdown by siRNA partially reversed the protective effects of 20C in rotenone-treated PC12 cells. Conclusion: The bibenzyl compound 20C protects PC12 cells from rotenone-induced apoptosis, at least in part, via activation of the Nrf2/ARE/HO-1 signaling pathway. PMID:27180985

  7. Neuroprotective effect of cannabidiol, a non-psychoactive component from Cannabis sativa, on beta-amyloid-induced toxicity in PC12 cells.

    PubMed

    Iuvone, Teresa; Esposito, Giuseppe; Esposito, Ramona; Santamaria, Rita; Di Rosa, Massimo; Izzo, Angelo A

    2004-04-01

    Abstract Alzheimer's disease is widely held to be associated with oxidative stress due, in part, to the membrane action of beta-amyloid peptide aggregates. Here, we studied the effect of cannabidiol, a major non-psychoactive component of the marijuana plant (Cannabis sativa) on beta-amyloid peptide-induced toxicity in cultured rat pheocromocytoma PC12 cells. Following exposure of cells to beta-amyloid peptide (1 micro g/mL), a marked reduction in cell survival was observed. This effect was associated with increased reactive oxygen species (ROS) production and lipid peroxidation, as well as caspase 3 (a key enzyme in the apoptosis cell-signalling cascade) appearance, DNA fragmentation and increased intracellular calcium. Treatment of the cells with cannabidiol (10(-7)-10(-4)m) prior to beta-amyloid peptide exposure significantly elevated cell survival while it decreased ROS production, lipid peroxidation, caspase 3 levels, DNA fragmentation and intracellular calcium. Our results indicate that cannabidiol exerts a combination of neuroprotective, anti-oxidative and anti-apoptotic effects against beta-amyloid peptide toxicity, and that inhibition of caspase 3 appearance from its inactive precursor, pro-caspase 3, by cannabidiol is involved in the signalling pathway for this neuroprotection.

  8. The anti-necrosis role of hypoxic preconditioning after acute anoxia is mediated by aldose reductase and sorbitol pathway in PC12 cells.

    PubMed

    Wu, Li-Ying; Ma, Zi-Min; Fan, Xue-Lai; Zhao, Tong; Liu, Zhao-Hui; Huang, Xin; Li, Ming-Ming; Xiong, Lei; Zhang, Kuan; Zhu, Ling-Ling; Fan, Ming

    2010-07-01

    It has been demonstrated that hypoxic preconditioning (HP) enhances the survival ability of the organism against the subsequent acute anoxia (AA). However, it is not yet clear whether necrosis induced by AA can be prevented by HP, and what are the underlying mechanisms. In this study, we examined the effect of HP (10% O(2), 48 h) on necrosis induced by AA (0% O(2), 24 h) in PC12 cells. We found that HP delayed the regulatory volume decrease and reduced cell swelling after 24 h of exposure to AA. Since aldose reductase (AR) is involved in cell volume regulation, we detected AR mRNA expression with reverse transcription-polymerase chain reaction (RT-PCR) techniques. The AR mRNA level was dramatically elevated by HP. Furthermore, an HP-induced decrease in cell injury was reversed by berberine chloride (BB), the inhibitor of AR. In addition, sorbitol synthesized from glucose catalyzed by AR is directly related to cell volume regulation. Subsequently, we tested sorbitol content in the cytoplasm. HP clearly elevated sorbitol content, while BB inhibited the elevation induced by HP. Further study showed that a strong inhibitor of sorbitol permease, quinidine, completely reversed the protection induced by HP after AA. These data provide evidence that HP prevents necrosis induced by AA and is mediated by AR and sorbitol pathway.

  9. Alterations in mitochondrial respiratory functions, redox metabolism and apoptosis by oxidant 4-hydroxynonenal and antioxidants curcumin and melatonin in PC12 cells

    SciTech Connect

    Raza, Haider John, Annie; Brown, Eric M.; Benedict, Sheela; Kambal, Amr

    2008-01-15

    Cellular oxidative stress and alterations in redox metabolisms have been implicated in the etiology and pathology of many diseases including cancer. Antioxidant treatments have been proven beneficial in controlling these diseases. We have recently shown that 4-hydroxynonenal (4-HNE), a by-product of lipid peroxidation, induces oxidative stress in PC12 cells by compromising the mitochondrial redox metabolism. In this study, we have further investigated the deleterious effects of 4-HNE on mitochondrial respiratory functions and apoptosis using the same cell line. In addition, we have also compared the effects of two antioxidants, curcumin and melatonin, used as chemopreventive agents, on mitochondrial redox metabolism and respiratory functions in these cells. 4-HNE treatment has been shown to cause a reduction in glutathione (GSH) pool, an increase in reactive oxygen species (ROS), protein carbonylation and apoptosis. A marked inhibition in the activities of the mitochondrial respiratory enzymes, cytochrome c oxidase and aconitase was observed after 4-HNE treatment. Increased nuclear translocation of NF-kB/p65 protein was also observed after 4-HNE treatment. Curcumin and melatonin treatments, on the other hand, maintained the mitochondrial redox and respiratory functions without a marked effect on ROS production and cell viability. These results suggest that 4-HNE-induced cytotoxicity may be associated, at least in part, with the altered mitochondrial redox and respiratory functions. The alterations in mitochondrial energy metabolism and redox functions may therefore be critical in determining the difference between cell death and survival.

  10. The role of sera from equine grass sickness on apoptosis induction in PC12 Tet-off p53 cell line

    PubMed Central

    Malekinejad, Hassan; Alizadeh-Tabrizi, Nazli; Ostadi, Araz; Fink-Gremmels, Johanna

    2015-01-01

    The pathogenesis of equine grass sickness (EGS) has not fully understood. A better understanding of the exact pathogenesis of diseases can help to make an accurate diagnosis. Previous studies reported some pathological damage of neuronal cells in EGS patients. In this study, primarily cytotoxicity of serum from three clinically EGS-diagnosed horses on PC12 Tet-off (PTO) cells was assessed. Subsequently, the apoptotic tests including cytochrome C release, caspase-3/7 activity measurement and DNA fragmentation assay were conducted to clarify the apoptotic effect of serum from EGS patients. Addition of serum from EGS patients at concentrations higher than 25% on PTO cells resulted in a significant cytotoxicity in Alamar blue reduction assay compared with serum from healthy horses. All three apoptotic endpoints showed that the serum from EGS patients does have capability to induce apoptosis. A remarkable up regulation of cytochrome C release accompanied with concentration- and time-dependent augmentation in caspase-3/7 activity and ultimately DNA fragmentation were observed. Our data suggest that serum from EGS patients might have potentially neurotoxic compounds, which exerts cytotoxic and apoptotic effects on neuronal cells. Moreover, the EGS serum-induced apoptosis attributes to augmentation of cytochrome C release and caspase-3/7 activity. PMID:25992246

  11. Protective Effect of L-Theanine on Cadmium-Induced Apoptosis in PC12 Cells by Inhibiting the Mitochondria-Mediated Pathway.

    PubMed

    Ben, Peiling; Zhang, Zhengping; Xuan, Chunxia; Sun, Shasha; Shen, Lei; Gao, Yanhong; Cao, Xiang; Zhou, Yi; Lan, Lei; Yin, Zhimin; Luo, Lan

    2015-08-01

    L-Theanine is an amino acid derivative from green tea. The present work was aimed at the effect of L-theanine on neuron-like rat pheochromocytoma (PC12) cells stimulated with cadmium chloride. Treatment with L-theanine before cadmium exposure increased cell viability; the experiments of Annexin V/PI staining indicated that L-theanine inhibited cadmium-induced cell apoptosis. Meanwhile, L-theanine decreased ROS production and protected from cadmium-induced disruption of mitochondrial transmembrane potential. Compared with cadmium-treated cells, L-theanine could also decrease the ratio of Bax/Bcl-2, as well as the level of cleaved caspase-9, caspase-3 and poly(ADP-ribose) polymerase. Furthermore, L-theanine depresses cadmium-induced up regulation of phosphorylations of PI3K/Akt, MAPK ERK1/2, and JNK signaling. These data suggest that L-theanine pretreatment reduces severity of cadmium toxicity probably via antioxidant action. Therefore, it may be concluded that L-theanine could be exploited for prevention of cadmium-induced diseases.

  12. The Induction of Serine/Threonine Protein Phosphorylations by a PDGFR/TrkA Chimera in Stably Transfected PC12 Cells*

    PubMed Central

    Biarc, Jordane; Chalkley, Robert J.; Burlingame, A. L.; Bradshaw, Ralph A.

    2012-01-01

    Stably transfected PC12 cells expressing a chimeric receptor composed of the extracellular domain of the platelet-derived growth factor receptor BB and the transmembrane and intracellular domains of TrkA, the nerve growth factor receptor, were stimulated for 20 min with platelet-derived growth factor and the resulting phosphoproteome was determined from affinity purified tryptic peptides identified by tandem MS (MS/MS) analyses. The changes in the levels of individual phosphorylation sites in stimulated cells versus control were ascertained by the stable isotope labeling of amino acids in cell culture technique. A total of 2035 peptides (806 proteins) were indentified and quantified in both data sets. Of these, 424 phosphopeptides on 259 proteins were found to be up-regulated and 392 sites on 206 proteins were down-regulated (1.8-fold or more). Protein kinases and phosphatases, as well as sites in many proteins involved in G-protein signaling, were prominently represented in the up-regulated group and more than half of the kinase up-regulated phosphosites could be clustered into three sequence motifs; a similar distribution was also found for the down-regulated sites. A comparison of the up-regulated motif profile observed to that calculated from a previous study of the EGFR-induced phosphoproteome in human HeLa cells at the same time point showed a considerable amount of similarity, supporting the view that RTK signal transduction pathways and downstream modifications are likely to be extensively overlapping. PMID:22027198

  13. Tumour necrosis factor-α suppresses the hypoxic response by NF-κB-dependent induction of inhibitory PAS domain protein in PC12 cells.

    PubMed

    Goryo, Kenji; Torii, Satoru; Yasumoto, Ken-Ichi; Sogawa, Kazuhiro

    2011-09-01

    Inflammation is often accompanied by hypoxia. However, crosstalk between signalling pathways activated by inflammation and signalling events that control adaptive response to hypoxia is not fully understood. Here we show that exposure to tumour necrosis factor-α (TNF-α) activates expression of the inhibitory PAS domain protein (IPAS) to suppress the hypoxic response caused by hypoxia-inducible factor (HIF)-1 and HIF-2 in rat pheochromocytoma PC12 cells but not in human hepatoma Hep3B cells. This induction of IPAS was dependent on the nuclear factor-κB (NF-κB) pathway and attenuated hypoxic induction of HIF-1 target genes such as tyrosine hydroxylase (TH) and vascular endothelial growth factor (VEGF). HIF-dependent reporter activity in hypoxia was also decreased following TNF-α treatment. Knockdown of IPAS mRNA by small interfering RNA (siRNA) restored the TNF-α-suppressed hypoxic response. These results indicate that TNF-α is a cell-type specific suppressor of HIFs and suggest a novel crosstalk between stimulation by inflammatory mediators and HIF-dependent hypoxic response.

  14. Reversal of Beta-Amyloid-Induced Neurotoxicity in PC12 Cells by Curcumin, the Important Role of ROS-Mediated Signaling and ERK Pathway.

    PubMed

    Fan, Cun-Dong; Li, Yuan; Fu, Xiao-Ting; Wu, Qing-Jian; Hou, Ya-Jun; Yang, Ming-Feng; Sun, Jing-Yi; Fu, Xiao-Yan; Zheng, Zun-Cheng; Sun, Bao-Liang

    2017-03-01

    Progressive accumulation of beta-amyloid (Aβ) will form the senile plaques and cause oxidative damage and neuronal cell death, which was accepted as the major pathological mechanism to the Alzheimer's disease (AD). Hence, inhibition of Aβ-induced oxidative damage and neuronal cell apoptosis by agents with potential antioxidant properties represents one of the most effective strategies in combating human AD. Curcumin (Cur) a natural extraction from curcuma longa has potential of pharmacological efficacy, including the benefit to antagonize Aβ-induced neurotoxicity. However, the molecular mechanism remains elusive. The present study evaluated the protective effect of Cur against Aβ-induced cytotoxicity and apoptosis in PC12 cells and investigated the underlying mechanism. The results showed that Cur markedly reduced Aβ-induced cytotoxicity by inhibition of mitochondria-mediated apoptosis through regulation of Bcl-2 family. The PARP cleavage, caspases activation, and ROS-mediated DNA damage induced by Aβ were all significantly blocked by Cur. Moreover, regulation of p38 MAPK and AKT pathways both contributed to this protective potency. Our findings suggested that Cur could effectively suppress Aβ-induced cytotoxicity and apoptosis by inhibition of ROS-mediated oxidative damage and regulation of ERK pathway, which validated its therapeutic potential in chemoprevention and chemotherapy of Aβ-induced neurotoxicity.

  15. Multi-porous electroactive poly(L-lactic acid)/polypyrrole composite micro/nano fibrous scaffolds promote neurite outgrowth in PC12 cells.

    PubMed

    Yu, Qiaozhen; Xu, Shuiling; Zhang, Kuihua; Shan, Yongming

    2013-01-05

    In this study, poly(L-lactic acid)/ammonium persulfate doped-polypyrrole composite fibrous scaffolds with moderate conductivity were produced by combining electrospinning with in situ polymerization. PC12 cells were cultured on these fibrous scaffolds and their growth following electrical stimulation (0-20.0 μA stimulus intensity, for 1-4 days) was observed using inverted light microscopy, and scanning electron microscopy coupled with the MTT cell viability test. The results demonstrated that the poly(L-lactic acid)/ammonium persulfate doped-polypyrrole fibrous scaffold was a dual multi-porous micro/nano fibrous scaffold. An electrical stimulation with a current intensity 5.0-10.0 μA for about 2 days enhanced neuronal growth and neurite outgrowth, while a high current intensity (over 15.0 μA) suppressed them. These results indicate that electrical stimulation with a moderate current intensity for an optimum time frame can promote neuronal growth and neurite outgrowth in an intensity- and time-dependent manner.

  16. TRICHLOROETHYLENE IHIBITS VOLTAGE-SENSITIVE CALCIUM CURRENTS IN DIFFERENTIATED PC 12 CELLS.

    EPA Science Inventory

    ABSTRACT BODY: It has been demonstrated recently that volatile organic compounds (VOCs)such as toluene, perchloroethylene and trichloroethylene inhibit function of voltage-sensitive calcium channels (VSSC). Such actions are hypothesized to contribute to the acute neurotoxicity of...

  17. Assessment of Antioxidant Activity and Neuroprotective Capacity on PC12 Cell Line of Frankenia thymifolia and Related Phenolic LC-MS/MS Identification

    PubMed Central

    Ben Mansour, Rim; Ksouri, Wided Megdiche; Cluzet, Stéphanie; Krisa, Stéphanie; Richard, Tristan

    2016-01-01

    This work aimed to investigate the richness of a Tunisian xerohalophyte Frankenia thymifolia aerial and root parts on phenolics and to evaluate the antioxidant and neuroprotective properties of this medicinal species. After fractionation using increasing and different solvent polarities, results displayed five fractions, where ethyl acetate (EtOAc) shoot and root fractions possess considerable total phenolic contents (221 and 308 mg of GAE/g of E, resp.) related to their important antioxidant activities such as ORAC (918 and 713 mg of TE/g of E), DPPH (282 and 821 mg of TE/g), and ABTS (778 and 1320 mg of TE/g) tests. Then, the identification of the main compounds by HPLC-DAD-ESI-MS and neuroprotective property of the most active fraction EtOAc were assessed. A total of 14 molecules were identified, which have been described for the first time in F. thymifolia. The major compounds identified were pinoresinol and kaempferol glycoside in aerial parts and gallic acid and ellagitannin in roots. Neuroprotective capacity against β-amyloid (Aβ) peptide induced toxicity in PC12 cells of EtOAc fraction showed a significant protective activity at lower concentration (25 and 50 µM). The relevant antioxidant and neuroprotective activities of F. thymifolia EtOAc fraction corroborated their chemical compositions. PMID:27829862

  18. [Effect of L-lysine alpha-oxidase from Trichoderma cf. aureoviride Rifai ВКМF-4268D on pheochromocytoma PC12 cell line].

    PubMed

    Lukasheva, E V; Ribakova, Yu S; Fedorova, T N; Makletsova, M G; Arinbasarova, A Yu; Medentzev, A G; Berezov, T T

    2015-01-01

    L-Amino acid oxidases (L-ААО, EC 1.4.3.2) comprise a group of flavoproteins, catalyzing oxidative deamination of L-alpha amino acids to the corresponding alpha-keto acids, NH3 and Н2О2. In most cases these enzymes present homodimeric molecules with a molecular mass of 100-150 kDa, which were shown to possess antiviral, antifungal and antitumor activity. L-lysine alpha-oxidase (LO) holds an outstanding place among this group of enzymes and its biological role may differ significantly from the other L-AAO, because it cleaves an essential amino acid - L-lysine without significant action on the other amino acids. Although much research has examined LO effects in the organism, the molecular basis of these effects is yet to be identified. To fill this gap, the present work addressed one of hypothetical mechanisms of LO biological action using the enzyme from Trichoderma cf. aureoviride Rifai ВКМF-4268D and rat pheochromocytoma PC-12 as a model cell line. Using flow cytometry a dose-dependent cytotoxicity of LO was shown. The significant growth of intracellular reactive oxygen species levels, detected by 2,7-dichlorodihydrofluorescein assay, implies generation of peroxide as one of the molecular mechanisms of LO cytotoxic action, although this does not rule out other probable ways of LO action in the organizm.

  19. Onjisaponin B Derived from Radix Polygalae Enhances Autophagy and Accelerates the Degradation of Mutant α-Synuclein and Huntingtin in PC-12 Cells

    PubMed Central

    Wu, An-Guo; Wong, Vincent Kam-Wai; Xu, Su-Wei; Chan, Wai-Kit; Ng, Choi-In; Liu, Liang; Law, Betty Yuen-Kwan

    2013-01-01

    Emerging evidence indicates important protective roles being played by autophagy in neurodegenerative disorders through clearance of aggregate-prone or mutant proteins. In the current study, we aimed to identify autophagy inducers from Chinese medicinal herbs as a potential neuroprotective agent that enhances the clearance of mutant huntingtin and α-synuclein in PC-12 cells. Through intensive screening using the green fluorescent protein-light chain 3 (GFP-LC3) autophagy detection platform, we found that the ethanol extracts of Radix Polygalae (Yuan Zhi) were capable of inducing autophagy. Further investigation showed that among three single components derived from Radix Polygalae—i.e., polygalacic acid, senegenin and onjisaponin B—onjisaponin B was able to induce autophagy and accelerate both the removal of mutant huntingtin and A53T α-synuclein, which are highly associated with Huntington disease and Parkinson disease, respectively. Our study further demonstrated that onjisaponin B induces autophagy via the AMPK-mTOR signaling pathway. Therefore, findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer, which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level. PMID:24248062

  20. Effects of c-Jun N-terminal kinase on Activin A/Smads signaling in PC12 cell suffered from oxygen-glucose deprivation.

    PubMed

    Wang, J Q; Xu, Z H; Liang, W Z; He, J T; Cui, Y; Liu, H Y; Xue, L X; Shi, W; Shao, Y K; Mang, J; Xu, Z X

    2016-02-29

    Activin A (Act A), a member of transforming growth factor-β (TGF-β) superfamily, is an early gene in response to cerebral ischemia. Growing evidences confirm the neuroprotective effect of Act A in ischemic injury through Act A/Smads signal activation. In this process, regulation networks are involved in modulating the outcomes of Smads signaling. Among these regulators, crosstalk between c-Jun N-terminal kinase (JNK) and Smads signaling has been found in the TGF-β induced epithelial-mesenchymal transition. However, in neural ischemia, the speculative regulation between JNK and Act A/Smads signaling pathways has not been clarified. To explore this issue, an Oxygen Glucose Deprivation (OGD) model was introduced to nerve-like PC12 cells. We found that JNK signal activation occurred at the early time of OGD injury (1 h). Act A administration suppressed JNK phosphorylation. In addition, JNK inhibition could elevate the strength of Smads signaling and attenuate neural apoptosis after OGD injury. Our results indicated a negative regulation effect of JNK on Smads signaling in ischemic injury. Taken together, JNK, as a critical site for neural apoptosis and negative regulator for Act A/Smads signaling, was presumed to be a molecular therapeutic target for ischemia.

  1. Stimulation of a Ca sup 2+ -dependent protein kinase by G sub M1 ganglioside in nerve growth factor-treated PC12 cells

    SciTech Connect

    Hilbush, B.S.; Levine, J.M. )

    1991-07-01

    The authors have investigated the ability of exogenous gangliosides to modulate nerve growth factor (NGF) signal transduction in PC12 cells. The effects of exogenous ganglioside G{sub M1} on multiple protein kinase activities were assayed by analyzing site-specific serine phosphorylation of tyrosine hydroxylase (TyrOHase) by two-dimensional phosphopeptide mapping. In the presence of NGF, exogenous G{sub M1} increased {sup 32}P incorporation into TyrOHase phosphopeptide T2, a Ca{sup 2+}/calmodulin-dependent protein kinase substrate whose phosphorylation is not normally affected by NGF treatment. In the absence of NGF, G{sub M1} treatment had no significant effects on TyrOHase phosphorylation. The removal of extracellular Ca{sup 2+} or blockade of dihydropyridine-sensitive Ca{sup 2+} channels prevented the G{sub M1}-induced increases in {sup 32}P incorporation into phosphopeptide T2. Exogenous G{sub M1} also potentiated K{sup +} depolarization-induced increases in the phosphorylation of TyrOHase. These results suggest that the stimulatory effects of exogenous G{sub M1} ganglioside on NGF actions may be due to its ability to potentiate a Ca{sup 2+}-dependent signaling pathway.

  2. Beneficial Effects of Ethanolic and Hexanic Rice Bran Extract on Mitochondrial Function in PC12 Cells and the Search for Bioactive Components.

    PubMed

    Hagl, Stephanie; Berressem, Dirk; Bruns, Bastian; Sus, Nadine; Frank, Jan; Eckert, Gunter P

    2015-09-11

    Mitochondria are involved in the aging processes that ultimately lead to neurodegeneration and the development of Alzheimer's disease (AD). A healthy lifestyle, including a diet rich in antioxidants and polyphenols, represents one strategy to protect the brain and to prevent neurodegeneration. We recently reported that a stabilized hexanic rice bran extract (RBE) rich in vitamin E and polyphenols (but unsuitable for human consumption) has beneficial effects on mitochondrial function in vitro and in vivo (doi:10.1016/j.phrs.2013.06.008, 10.3233/JAD-132084). To enable the use of RBE as food additive, a stabilized ethanolic extract has been produced. Here, we compare the vitamin E profiles of both extracts and their effects on mitochondrial function (ATP concentrations, mitochondrial membrane potential, mitochondrial respiration and mitochondrial biogenesis) in PC12 cells. We found that vitamin E contents and the effects of both RBE on mitochondrial function were similar. Furthermore, we aimed to identify components responsible for the mitochondria-protective effects of RBE, but could not achieve a conclusive result. α-Tocotrienol and possibly also γ-tocotrienol, α-tocopherol and δ-tocopherol might be involved, but hitherto unknown components of RBE or a synergistic effect of various components might also play a role in mediating RBE's beneficial effects on mitochondrial function.

  3. High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells.

    PubMed

    Pezzati, R; Bossi, M; Podini, P; Meldolesi, J; Grohovaz, F

    1997-08-01

    The calcium pools segregated within the endoplasmic reticulum, Golgi complex, exocytic, and other organelles are believed to participate in the regulation of a variety of cell functions. Until now, however, the precise intracellular distribution of the element had not been established. Here, we report about the first high-resolution calcium mapping obtained in neurosecretory PC12 cells by the imaging mode of the electron energy loss spectroscopy technique. The preparation procedure used included quick freezing of cell monolayers, followed by freeze-drying, fixation with OSO4 vapors, resin embedding, and cutting of very thin sections. Conventional electron microscopy and high-resolution immunocytochemistry revealed a high degree of structural preservation, a condition in which inorganic elements are expected to maintain their native distribution. Within these cells, calcium signals of nucleus, cytosol, and most mitochondria remained below detection, whereas in other organelles specific patterns were identified. In the endoplasmic reticulum, the distribution was heterogeneous with strongly positive cisternae (more often the nuclear envelope and stacks of parallel elements that are frequent in quick frozen preparations) lying in the proximity of or even in direct continuity with other, apparently negative cisternae. The Golgi complexes were labeled strongly and uniformly in all cisternae and part of their vesicles, with no appreciable differences along the cis-trans axis. Weaker or negative signals were recorded from the trans-Golgi network elements and from scattered vesicles, whereas in contrast secretion granules were strongly positive for calcium. These results are discussed in relation to the existing knowledge about the mechanisms of calcium transport in the variations organelles, and about the processes and functions regulated by organelle lumenal calcium in eukaryotic cells.

  4. Yizhijiannao Granule and a combination of its effective monomers, icariin and Panax notoginseng saponins, inhibit early PC12 cell apoptosis induced by beta-amyloid (25–35)☆

    PubMed Central

    Zhang, Ting; Zhang, Zhanwei; Dong, Keli; Li, Guangcheng; Zhu, Hong

    2012-01-01

    One of our previous studies showed that Yizhijiannao Granule, a compound Chinese medicine, effectively improved the clinical symptoms of Alzheimer's disease. In the present study, we established a model of Alzheimer's disease using beta-amyloid (25–35) in PC12 cells, and treated the cells with Yizhijiannao Granule and its four monomers, i.e., icariin, catechin, Panax notoginseng saponins, and eleutheroside E. Flow cytometry showed that Yizhijiannao Granule-containing serum, icariin, Panax notoginseng saponins, and icariin + Panax notoginseng saponins were protective against beta-amyloid (25–35)-induced injury in PC12 cells. Icariin in combination with Panax notoginseng saponins significantly inhibited early apoptosis of PC12 cells with beta-amyloid (25–35)-induced injury compared to icariin or Panax notoginseng saponins alone. The effects of icariin + Panax notoginseng saponins were similar to the effects of Yizhijiannao Granule. The findings indicate that two of the effective monomers of Yizhijiannao Granule, icariin and Panax notoginseng saponins, can synergistically inhibit early apoptosis of PC12 cells induced by beta-amyloid (25–35). PMID:25624809

  5. Agomelatine and duloxetine synergistically modulates apoptotic pathway by inhibiting oxidative stress triggered intracellular calcium entry in neuronal PC12 cells: role of TRPM2 and voltage-gated calcium channels.

    PubMed

    Akpinar, Abdullah; Uğuz, Abdülhadi Cihangir; Na